β-cyclodextrin compositions, and use to prevent transmission of sexually transmitted diseases

ABSTRACT

Methods of reducing the risk of transmission of a sexually transmitted pathogen by contacting the pathogen or cells susceptible to infection by the pathogen with a β-cyclodextrin are provided. Methods for reducing the risk of transmission of a sexually transmitted pathogen to or from a subject by contacting the pathogen or cells susceptible to the pathogen in the subject with a pharmaceutical composition containing a β-cyclodextrin also are provided. Accordingly, pharmaceutical compositions, which include 1) a β-cyclodextrin, which is in an amount that blocks passage of the pathogen through lipid rafts in the membrane of a cell susceptible to the pathogen, and 2) a contraceptive, an agent for treating a sexually transmitted disease, a lubricant, or a combination thereof, are provided, as are composition formulated from a solid substrate that contains an amount of β-cyclodextrin useful for reducing the risk of transmission of a sexually transmitted pathogen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/025,661 filed Dec. 28, 2004, now U.S. Pat. No. 7,202,231; whichis a continuation application of U.S. application Ser. No. 09/802,779filed Mar. 8, 2001, now issued as U.S. Pat. No. 6,835,717; which is acontinuation-in-part application of U.S. application Ser. No. 09/801,393filed Mar. 7, 2001, now abandoned; which claims the benefit under 35 USC§ 119(e) to U.S. application Ser. No. 60/267,199 filed Feb. 7, 2001, nowabandoned and to U.S. Application Ser. No. 60,187,784 filed Mar. 8,2000, now abandoned. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

GRANT INFORMATION

This invention was made in part with government support under Grant Nos.AI31806 and AI4629 awarded by the National Institutes of Health andGrant No. HD39613 awarded by the U.S. Public Health Service. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to agents and methods for preventing aviral or microbial infection and, more specifically, to compositionscontaining a β-cyclodextrin and methods of using such compositions toreduce the risk of transmission of a sexually transmitted disease.

2. Background Information

Sexually transmitted diseases (STDs) are among the most common types ofinfections. Three bacterial STDs—gonorrhea, chlamydial infections, andsyphilis—are particularly common, and account for a great deal ofmorbidity, including infertility, ectopic pregnancy, and loss ofproductivity (see Harrison's “Principles of Internal Among the viralSTDs, human papilloma virus and hepatitis B virus are among the mostcommon, and are associated with cervical carcinoma and hepatocellularcarcinoma, respectively.

In the past couple of decades, acquired immunodeficiency disease (AIDS)associated with sexual transmission of human immunodeficiency virus(HIV) has emerged as a global health threat. In industrializedcountries, education as to the use of condoms and the practice of “safesex” reduced the levels of new HIV infection and of AIDS deathsfollowing a peak in the mid-1990's. However, the decreased number ofAIDS deaths and the availability of medications that appear to increasethe life spans of AIDS patients may have created a false sense ofsecurity, and it now appears that this trend may reverse. In manynon-industrialized countries, AIDS is an epidemic, and it is notinconceivable that millions may die from this disease in the next fewyears.

HIV can be transmitted in a number of ways, including throughcontaminated blood products, and from mother to offspring duringgestation, child birth or breast feeding. However, newly acquired HIVinfections are largely the result of sexual contact, particularlyheterosexual contact. A number of factors appear to determine whetherHIV is transmitted sexually, including the type of sex act,susceptibility of the exposed partner, infectivity of the infectedpartner, and the biological properties of the particular HIV subtype.

Prevention of the spread of HIV infection requires interventions of boththe infected and uninfected populations. In particular, since only asmall percentage of HIV-infected individuals are aware of their carrierstatus, a significant prevention effort must be made by the susceptiblepopulation. Mechanical barriers such as condoms can be effective inpreventing sexual transmission of HIV. However, this method is notalways accepted by male partners, and can be impractical for use bywomen. Topical microbicides currently available have proven inadequate,and the widely used surfactant microbicide, nonoxynol-9, which is usedas a spermicide, may actually increase HIV infection by inducing genitalulcerations. Thus, in the absence of an effective vaccine, otherbiomedical methods must be identified, particularly those that can bepracticed by the susceptible population.

Semen from HIV infected men and cervical mucus from HIV infected womencontain free virus as well as HIV-infected cells and, sexualtransmission of HIV may occur due to both forms of the virus. Thus, aneed exists for a therapeutic agent that reduces or eliminatestransmission of free HIV as well as cell-associate virus infection,thereby reducing the risk of transmission of HIV and other sexuallytransmitted pathogens. The present invention satisfies this need andprovides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to methods of reducing the risk oftransmission of a sexually transmitted pathogen, includingcell-associated and cell-free sexually transmitted pathogens. In oneembodiment, a method of the invention is performed by contacting thepathogen or cells susceptible to infection by the pathogen with aβ-cyclodextrin (βCD). The pathogen can be any pathogen involved in theetiology of a sexually transmitted disease, particularly a pathogen thatinfects a susceptible cell through contact with lipid rafts in themembrane of the cell. As such, the pathogen can be an enveloped virus,for example, an immunodeficiency virus such as human immunodeficiencyvirus (HIV), a T lymphotrophic virus such as human T lymphotrophic virus(HTLV), a herpesvirus such as a herpes simplex virus (HSV), a measlesvirus, or an influenza virus. The pathogen also can be a microbialpathogen, for example, a bacterium, a yeast such as a Candida spp., amycoplasma, a protozoan such as a Trichomona spp., or a Chlamydia spp.The βCD can be any βCD derivative, for example,2-hydroxypropyl-β-cyclodextrin.

In another embodiment, a method of the invention provides a means toreduce the risk of transmission of a sexually transmitted pathogen to orfrom a subject, which can be any subject susceptible to a sexuallytransmitted disease, for example, a vertebrate, particularly a mammal,including a human. Such a method can be performed, for example, bycontacting the pathogen or cells susceptible to infection by thepathogen in the subject with a pharmaceutical composition comprising aβ-cyclodextrin (βCD), thereby reducing the risk of the subject becominginfected with the sexually transmitted the pathogen. Such a method alsocan be performed, for example, by contacting the pathogen or cellssusceptible to infection by the pathogen in a subject having a sexuallytransmitted disease with a pharmaceutical composition comprising a βCD,thereby reducing the risk of transmission of the sexually transmitteddisease by the subject.

he cells susceptible to infection by the pathogen can be any cellsdepending, in part, on the pathogen, including epithelial cells,particularly vaginal epithelial cells or rectal epithelial cells.Furthermore, the cells susceptible to infection, as well as the pathogenin a cell-free form, can be present in a secretion produced by thesubject, for example, in semen or in a vaginal secretion. Thepharmaceutical composition can be formulated in a solution, a gel, afoam, an ointment, a cream, a paste, a spray, or the like; or can beformulated as a component of a suppository, a film, a sponge, a condom,a bioadhesive polymer, a diaphragm, or the like; and can contain, inaddition to the βCD, one or more agents useful to a sexually activesubject, for example, a contraceptive, an antimicrobial or antiviralagent, a lubricant, or a combination thereof.

The present invention also relates to a pharmaceutical composition,which includes 1) βCD, which is in an amount that blocks passage of thepathogen through lipid rafts in the membrane of a cell susceptible tothe pathogen, and 2) a contraceptive, an antimicrobial or antiviralagent, a lubricant, or a combination thereof. A contraceptive useful ina pharmaceutical composition of the invention can be any contraceptive,for example, a spermicide. Similarly, an antimicrobial or antiviralagent for treating a sexually transmitted disease can be any agent thatgenerally is used to treat or prevent infection by a sexuallytransmitted pathogen, or an opportunistic pathogen associated with asexually transmitted disease, including, for example, an antibiotic.

The present invention further relates to a composition, comprising asolid substrate that contains an amount of βCD useful for reducing therisk of transmission of a sexually transmitted pathogen. The solidsubstrate can be a barrier, which is composed of a relativelyimpermeable substrate, for example, a condom, diaphragm, vaginal film orglove, which contains the βCD at least on its surface; or can becomposed of an absorptive material, for example, a sponge or a tampon,which contains the β-cyclodextrin incorporated therein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of reducing the risk oftransmission of a sexually transmitted pathogen. A method of theinvention is based, in part, on the determination that entry of asexually transmitted pathogen, for example, a sexually transmittedenveloped virus, into a cell depends, at least in part, on the presenceof lipid rafts in the membranes of cells susceptible to the pathogen,and the determination that contact of such susceptible cells or of thepathogen with a β-cyclodextrin, which disrupts the structure of lipidrafts, blocks the ability of the pathogen to infect an otherwisesusceptible cell.

β-cyclodextrins (βCDs) are widely used as solubilizing agents,stabilizers, and inert excipients in pharmaceutical compositions (seeU.S. Pat. Nos. 6,194,430; 6,194,395; and 6,191,137, each of which isincorporated herein by reference). βCDs are cyclic compounds containingseven units of I-(1→4) linked D-glucopyranose units, and act ascomplexing agents that can form inclusion complexes and have concomitantsolubilizing properties (see U.S. Pat. No. 6,194,395; see, also,Szejtli, J. Cyclodextrin Technol. 1988). As disclosed herein, βCDs alsocan block passage of a sexually transmitted pathogen through themembrane of a susceptible cell by disrupting the lipid rafts in cellmembrane.

The compositions and methods of the invention are exemplified using2-hydroxypropyl-βCD (2-OH-βCD). However, any βCD derivative can be usedin a composition or method of the invention, provided the βCD derivativedisrupts lipid rafts in the membranes of cells susceptible to a sexuallytransmitted pathogen without causing undesirable side effects (seeExample 3). βCDs act, in part, by removing cholesterol from cellmembranes, and different βCDs are variably effective in such removal.For example, methyl-βCD removes cholesterol from cell membranes veryefficiently and quickly and, as a result, can be toxic to cells, whichrequire cholesterol for membrane integrity and viability. In comparison,a βCD derivative such as 2-OH-βCD can effectively remove cholesterolfrom cells without producing undue toxicity. Thus, it will be recognizedthat a βCD useful in a composition or method of the invention is onethat removes cholesterol in an amount that disrupts lipid rafts, withoutsubstantially reducing cell viability (see, for example, Rothblat andPhillips, J. Biol. Chem. 257:4775-4782, 1982, which is incorporatedherein by reference).

βCDs useful in the present invention include, for example, βCDderivatives wherein one or more of the hydroxy groups is substituted byan alkyl, hydroxyalkyl, carboxyalkyl, alkylcarbonyl, carboxyalkoxyalkyl,alkylcarbonyloxyalkyl, alkoxycarbonylalkyl or hydroxy-(mono orpolyalkoxy)alkyl group or the like; and wherein each alkyl or alkylenemoiety contains up to about six carbons. Substituted βCDs that can beused in the present invention include, for example, polyethers (see, forexample, U.S. Pat. No. 3,459,731, which is incorporated herein byreference); ethers, wherein the hydrogen of one or more βCD hydroxygroups is replaced by C1 to C6 alkyl, hydroxy-C1-C6-alkyl, carboxy-C1-C6alkyl, C1-C6 alkyloxycarbonyl-C1-C6 alkyl groups, or mixed ethersthereof. In such substituted βCDs, the hydrogen of one or more βCDhydroxy group can be replaced by C1-C3 alkyl, hydroxy-C2-C4 alkyl, orcarboxy-C1-C2 alkyl, for example, by methyl, ethyl, hydroxyethyl,hydroxypropyl, hydroxybutyl, carboxymethyl or carboxyethyl. It should berecognized that the term “C1-C6 alkyl” includes straight and branchedsaturated hydrocarbon radicals, having from 1 to 6 carbon atoms.Examples of βCD ethers include dimethyl-βCD. Examples of βCD polyethersinclude hydroxypropyl-p-βCD and hydroxyethyl-βCD (see, for example,Nogradi, “Drugs of the Future” 9(8):577-578, 1984; Chemical andPharmaceutical Bulletin 28: 1552-1558, 1980; Yakugyo Jiho No. 6452 (Mar.28, 1983); Angew. Chem. Int. Ed. Engl. 19: 344-362, 1980; U.S. Pat. No.3,459,731; EP-A-0,149,197; EP-A-0,197,571; U.S. Pat. No. 4,535,152;WO-90/12035; GB-2,189,245; Szejtli, “Cyclodextrin Technology” (KluwerAcademic Publ. 1988); Bender et al., “Cyclodextrin Chemistry”(Springer-Verlag, Berlin 1978); French, Adv. Carb. Chem. 12:189-260;Croft and Bartsch, Tetrahedron 39:1417-1474, 1983; Irie et al., Pharm.Res. 5:713-716, 1988; Pitha et al., Internat'l. J. Pharm. 29:73, 1986;U.S. Pat. No. 5,134,127 A; U.S. Pat. Nos. 4,659,696 and 4,383,992, eachof which is incorporated herein by reference; see, also, U.S. Pat. No.6,194,395).

In one embodiment, a method of the invention is performed by contactingthe pathogen or cells susceptible to infection by the pathogen with aβCD. As used herein, reference to cells being “susceptible” to infectionby a sexually transmitted pathogen means that the membranes of the cellscontain lipid rafts, to which the pathogen can associate and throughwhich it can traverse the membrane. Cells susceptible to a sexuallytransmitted pathogen are exemplified by vaginal epithelial cells, whichcontain lipid rafts that are used by HIV to traverse the cell membrane(see Example 1).

As used herein, the term “sexually transmitted pathogen” refers to anyviral or microbial organism that causes a sexually transmitted disease.The term “sexually transmitted disease” refers to a disease that istransmitted through sexual contact with an infected individual. Sexuallytransmitted diseases and the sexually associated pathogens associatedtherewith are well known in the art and include, for example, thosecaused by bacteria such as gonorrhea (Neisseria gonnorrhoeae) andsyphilis (Treponema pallidum), and infections due to Chlamydia spp. suchas C. trachomatis, Calymmatobacterium granulomatis, Ureaplasmaurealyticum, Mycoplasma hominus, Gardnerella vaginalis, and Group BStreptococcus spp.; those caused by viruses such AIDS (HIV-1 and HIV-2),genital herpes (Herpes simplex type 2; HSV-2), and infections due tohuman T lymphotrophic virus type I (HTLV-1), human papillomaviruses,Cytomegalovirus, Molluscum contagiosum virus, hepatitis B virus, andpossibly HSV-1, HTLV-II, and Epstein-Barr virus; and those due to yeastsuch as Candida spp., for example, C. albicans, or to protozoans such asTrichomona spp., for example, T vaginalis (see Harrison's “Principles ofInternal Medicine”, supra, 1994).

As disclosed herein, where a sexually transmitted disease is due toinfection by a pathogen that traverses a susceptible cell through lipidrafts in the membrane of the cell, the risk of transmission of thepathogen can be reduced by contacting the pathogen or the cell with aβCD (see Examples 2 to 4). As used herein, the term “reducing the riskof transmission of a sexually transmitted pathogen” means that thelikelihood that the pathogen will infect a susceptible cell is decreaseddue to contact of the pathogen or the cell with a βCD as compared to thelikelihood of infection of the cell in the absence of βCD treatment. Thelikelihood of infection of such cells (i.e., untreated or contacted witha βCD) can be determined by examining populations of such cells anddetermining the levels of infection of the cells by the pathogen usingmethods as disclosed herein or otherwise known in the art (see Examples2 to 4).

A method of the invention is performed, for example, by contacting thepathogen or cells susceptible to infection by the pathogen with a βCD.As used herein, the term “contacting,” when used in reference to a βCDand the pathogen or cells susceptible to a sexually transmittedpathogen, means that the βCD is placed in sufficient proximity to thepathogen or the susceptible cells such that it prevents the pathogenfrom entering a cell through lipid rafts or such that it disrupts lipidrafts in the membranes of the susceptible cells. Thus, the βCD can beadded to cells in culture, for example, thereby contacting the cellswith the βCD; or can be inserted into vagina or rectum of a subjecteither in a liquid or liquid-like form such as a gel, foam, or the like,or as a suppository, or in combination with a solid substrate such as acondom, thereby contacting the sexually transmitted pathogen or thecells susceptible to the pathogen in vivo.

The significance of detergent-insoluble, glycolipid-enriched membranedomains (“lipid rafts”) has been demonstrated, particularly in regard toactivation and signaling in T lymphocytes. Lipid rafts can be viewed asfloating rafts composed of sphingolipids and cholesterol that sequesterglycosylphosphatidylinositol-(GPI)-linked proteins such as Thy-1 andCD59. CD45, a 200 kDa transmembrane phosphatase protein, is excludedfrom these domains. Human immunodeficiency virus type 1 (HIV-1)particles produced by infected T cell lines acquire the GPI-linkedproteins Thy-1 and CD59, as well as the ganglioside GM1, which is knownto partition preferentially into lipid rafts. In contrast, despite itshigh expression on the cell surface, CD45 is poorly incorporated intovirus particles. Confocal fluorescence microscopy revealed that HIV-1proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specificfluorescent lipid, DiIC16 (see below), in uropods of infected Jurkatcells. CD45 did not colocalize with HIV-1 proteins and was excluded fromuropods. Dot immunoassay of TRITON X-100 detergent-extracted membranefractions revealed that HIV-1 p17 matrix protein and gp41 were presentin the detergent-resistant fractions and that (³H)-myristic acid-labeledHIV Gag showed a nine-to-one enrichment in lipid rafts. As disclosedherein, the budding of HIV virions through lipid rafts is associatedwith the presence of host cell cholesterol, sphingolipids, andGPI-linked proteins within these domains in the viral envelope,indicating preferential sorting of HIV Gag to lipid rafts (see Example1).

Glycolipid-enriched membrane (GEM) domains are organized areas on thecell surface enriched in cholesterol, sphingolipids, and GPI-linkedproteins. These domains have been described as “rafts” that serve asmoving platforms on the cell surface (Shaw and Dustin, Immunity6:361-369, 1997). The domains, now referred to as “lipid rafts,” existin a more ordered state, conferring resistance to TRITON X-100 detergenttreatment at 4° C. (Schroeder et al., J. Biol. Chem. 273:1150-1157,1998). Many proteins are associated with lipid rafts, includingGPI-linked proteins, Src family kinases, protein kinase C, actin andactin-binding proteins, heterotrimeric and small G proteins, andcaveolin (see, for example, Arni et al., Biochem. Biophys. Res. Commun.225:8001-807, 1996; Cinek and Horejsi, J. Immunol. 149:2262-2270, 1992;Robbins et al., Mol. Cell. Biol. 15:3507-3515, 1995; and Sargiacomo etal., J. Cell. Biol. 122:789-807, 1993). Saturated acyl chains of the GPIanchor have been shown to be a determinant for the association ofGPI-linked proteins with lipid rafts (Rodgers et al., Mol. Cell. Biol.14:5384-5391, 1994; Schroeder et al., Proc. Natl. Acad. Sci., USA91:12130-12134, 1994). Lipid rafts exclude certain transmembranemolecules, specifically the membrane phosphatase CD45 (Arne et al.,supra, 1996; Rodgers and Rose, J. Cell. Biol.135:1515-1523, 1996).Exclusion of CD45 results in the accumulation of phosphorylatedsignaling molecules in lipid rafts, and T cell activation requiresclustering of signaling molecules in these membrane domains(Lanzavecchia et al., Cell 96:1-4, 1999).

HIV-1 excludes CD45 from its membrane, despite the abundance of CD45 onthe cell surface. This result was in contrast to that observed for othermembrane proteins, some of which are expressed at lower levels thanCD45, but were efficiently incorporated by the virus (Orentas andHildreth, AIDS Res. Hum. Retrovir. 9:1157-1165, 1993, which isincorporated herein by reference). CD45 is a large, heavilyglycosylated, multiply spliced transmembrane protein that has twocytoplasmic tyrosine phosphatase domains. Extracellularly, it may extendas much as 40 nm from the cell surface, while intracellularly it has alarge cytoplasmic tail of 707 amino acids. CD45 is one of the mosthighly expressed leukocyte surface proteins, and as much as 10 to 25% ofthe lymphocyte cell surface can be covered with CD45. If HIV-1incorporated host proteins in a random manner, a significant number ofCD45 molecules should be present on the virus.

As disclosed herein, CD45 is excluded from HIV-1 particles as a resultof virus budding from lipid rafts, which also exclude CD45 (Example 1).HIV-1 incorporates the lipid raft-specific ganglioside, GM1, as well asGPI-linked proteins Thy-1 and CD59. Confocal fluorescence microscopyshowed that viral proteins colocalize with Thy-1, CD59, GM1, and1,19-dihexadecyl-3,3,39,39-tetramethyl indocarbocyanine (DiIC16; Arthuret al., Science 258:1935-1938, 1992, which is incorporated herein byreference), a fluorescent dye that partitions to ordered domains inuropods on infected cells. In contrast, CD45 is excluded from theseGPI-linked protein-rich membrane projections. Upon membranefractionation, HIV matrix (MA) and gp41, the transmembrane subunit ofenvelope (Env), are present in detergent-resistant, GPI-linkedprotein-rich fractions, confirming their association with lipid rafts.Specifically, myristylated Gag localizes predominantly to the detergentresistant lipid rafts. These results indicate that HIV-1 budding occursthrough lipid rafts, thereby accounting for the cholesterol-rich,sphingolipid-rich virus membrane, which bears GPI-linked proteins suchas Thy-1 and CD59, but lacks CD45.

Lipid raft-associated molecules, including the GPI-anchored proteinsThy-1 and CD59 and the ganglioside GM1, colocalized with HIV-1 proteinson the cell surface as determined by confocal fluorescence microscopy(Example 1). Virus phenotyping with MAbs also indicated that thesemolecules were incorporated into HIV-1 particles. In contrast, CD45 wasexcluded from HIV-1 protein-rich uropods and was also excluded from theviral membrane. Similarly, DiIC16 colocalized with HIV-1 proteins, whileDiIC12, a lipid analog that prefers fluid membrane domains, was excludedfrom these areas. Dot blot immunoassays of membrane fractions confirmedthe presence of HIV-1 gp41 and MA proteins in lipid rafts, and labelingof cells with tritiated myristic acid and immunoprecipitation showed thepartitioning of myristylated Gag to lipid rafts.

It was previously reported that HIV-1 acquires CD55 (DAF) and CD59,which inhibit steps in the complement pathway (Marschang et al., Eur. J.Immunol. 25:285-290, 1995; Saifuddin et al., J. Gen. Virol.78:1907-1911, 1997). CD55 and CD59 are GPI-linked proteins that areenriched in GEM domains and, together, provide an advantage for thevirus by shielding it from lysis and from neutralization by complement.The results disclosed herein confirm and extend the previousobservations by demonstrating that HIV-1 incorporates GPI-anchoredproteins, which preferentially sort to lipid rafts, and that lipid raftsare the cell membrane microdomains from which HIV-1 buds (Example 1).The high concentration of cholesterol and sphingolipids in lipid raftsexplains the high levels of these lipids in the membrane of HIV-1 andsupports this model of HIV-1 budding. Interestingly, inhibition ofcholesterol synthesis decreases the production of virus from infectedcells (Maziere et al., Biomed. Pharmacother. 48:63-67, 1994). Since itis unlikely that viral proteins can aggregate individual cholesterol andsphingolipid molecules, the Gag (MA) protein may preferentially interactwith existing lipid rafts, where aggregation of Gag (MA) molecules caninitiate virus budding. In this manner, sphingolipid-rich andcholesterol-rich lipid rafts can be efficiently taken up by new virusesduring budding.

The role of lipid rafts in viral infection can further be extended toviruses other than HIV. For example, selective budding occurs for aninfluenza virus, fowl plague virus, from ordered lipid domains(Scheiffele et al., J. Biol. Chem. 274:2038-2044, 1999, which isincorporated herein by reference). The requirement for cholesterol andsphingolipids in target membranes for Semliki Forest virus fusion alsohas been established (Nieva et al., EMBO J. 13:2797-2804, 1994; Phalenand Kielian, J. Cell Biol. 112:615-623, 1991, each of which isincorporated herein by reference). The interactions of lipid rafts withaccessory HIV-1 molecules such as Vif and Nef can have important rolesin virus budding, since interactions of myristylated HIV and simianimmunodeficiency virus Nef with Lck, which is present in lipid rafts,and its incorporation into virions have been established (see, forexample, Collette et al., J. Biol. Chem. 271:6333-6341, 1996; Flahertyet al., AIDS Res. Hum. Retrovir. 14:163-170, 1998).

The incorporation of Thy-1, CD59, and other GPI-linked proteins into theviral envelope can have a number of consequences for virus infection andpathogenicity. For example, Thy-1, CD59, and CD55 have cell-signalingcapabilities, and the transfer of these highly concentrated proteinsinto the host cell by HIV-1 particles can be involved in triggering anactivation signal leading to interleukin-2 production and T cellproliferation. GPI-linked proteins are physically associated with theI-subunit of G proteins, which are important in signal transduction,while other signaling molecules, such as Src family kinases, areassociated with lipid rafts (see, for example, Rodgers et al., Mol.Cell. Biol. 14:5384-5391, 1994). Delivery of these signal transductionmolecules to the host cells by the virus can have important effects onvirus infectivity, depending, for example, on the cell type and itsstate of activation. Among other effects, GPI-linked molecules actingthrough G proteins can activate integrins such as LFA-1, which cancontribute greatly to HIV-1 infectivity and syncytium formation (seeGomez and Hildreth, J. Virol. 69:4628-4632, 1995).

A recent model suggests that CD45 is driven out of cap sites that serveas zones for cellular adhesion and activation between a T cell and anantigen-presenting cell (Shaw and Dustin, Immunity 6:361-369, 1997). Inthis model, short, low-affinity molecules such as the T cell receptorare clustered into the cap site, enhancing the two-dimensional affinityof these molecules for their ligands. This same mechanism results inexclusion of CD45 and capping of GPI-linked proteins and lipid raftsinto the areas of cell-to-cell contact. Viral protein targeting throughassociation with lipid rafts into cap sites may facilitate virusparticle formation at that site on the surface by directing myristylatedmatrix proteins and accessory molecules.

The exclusion of CD45 from virions may be an important aspect of HIVassembly. Since the cytoplasmic tail of CD45 is so large (more than 700amino acids), incorporation of CD45 can hinder critical interactionsbetween gp41 and matrix proteins or other molecules. Furthermore, thelong, highly negatively charged extracellular domain of CD45, determinedto be as long as 41 nm, can sterically hinder viral binding to targetcells if it were to be incorporated, considering that a virus particleis only about 100 nm in diameter.

The results disclosed in Example 1 indicate that HIV-1 buds throughlipid rafts. During the course of infection, the cell becomes activatedand polarization occurs, capping normally dispersed lipid rafts alongwith GPI-linked proteins and associated intracellular signalingmolecules, and membrane areas containing CD45 can be cleared out of thecap site. The newly translated viral Gag precursor protein associatedwith lipid rafts then can be directed to the capped pole, where assemblyand budding occurs. Palmitylated gp41 (gp160) is also directed intolipid rafts, and the interaction of its cytoplasmic tail with MA inlipid rafts can prevent its internalization, allowing for theincorporation of gp160 into virions only at the site of budding (seeEgan et al., J. Virol. 70:6547-6556, 1996; Yu et al., J. Virol.66:4966-4971, 1992). Individual targeting of Gag and Env to the samesite at the membrane can be an important means for delivering theseproteins to the site of budding, since Gag and Env are processed andtransported in different pathways within the cell. The host membranethen can become the new viral coat, resulting in the incorporation ofcholesterol, sphingolipids, Thy-1, and CD59 and in the exclusion ofCD45. HIV-1 also acquires functional adhesion molecules from host cells(Orentas and Hildreth, supra, 1993). These host-acquired proteins cansignificantly affect the biology of HIV-1 (see, for example, Fortin etal., J. Virol. 71:3588-3596, 1997).

As described above, budding of HIV-1 particles occurs at lipid rafts,which are characterized by a distinct lipid composition that includeshigh concentrations of cholesterol, sphingolipids, and glycolipids.Since cholesterol plays a key role in the entry of some other viruses,the role in HIV-1 entry of cholesterol and lipid rafts in the plasmamembrane of susceptible cells was investigated (Example 2). A βCDderivative, 2-hydroxypropyl-β-cyclodextrin (2-OH-βCD), was used todeplete cellular cholesterol and disperse lipid rafts. As disclosedherein, removal of cellular cholesterol rendered primary cells and celllines highly resistant to HIV-1-mediated syncytium formation and toinfection by both CXCR4- and CCR5-specific viruses. 2-OH-βCD treatmentof the virus or cells partially reduced HIV-1 binding, while renderingchemokine receptors highly sensitive to antibody-mediatedinternalization, but had no effect on CD4 expression. These effects werereadily reversed by incubating cholesterol-depleted cells with lowconcentrations of cholesterol-loaded 2-OH-βCD to restore cholesterollevels. Cholesterol depletion also made cells resistant to SDF-1-inducedbinding to ICAM-1 through LFA-1. This may have contributed to thereduction in HIV-1 binding to cells after treatment with the βCD, sinceLFA-1 contributes significantly to cell binding by HIV-1 which, likeSDF-1α, can trigger CXCR4 function through gp120. These results indicatethat cholesterol is involved in the HIV-1 co-receptor function ofchemokine receptors and is required for infection of cells by HIV-1 (seeExample 2).

As discussed above, cholesterol, sphingolipids, and GPI-anchoredproteins are enriched in lipid rafts (see Simons and Ikonen, Nature387:569-572, 1997). The high concentration of cholesterol andsphingolipids in lipid rafts results in a tightly packed, ordered lipiddomain that is resistant to non-ionic detergents at low temperature. Thestructural protein caveolin causes formation of flask-like invaginations(caveolae) in the cell membrane with a lipid composition very similar tothat of lipid rafts (Schnitzer et al., Science 269:1435-1439, 1995).Signaling molecules, including Lck, LAT, NOS, and G protein I subunit,are localized to rafts on the intracellular side of the membrane, andare targeted by lipid modifications such as palmitylation,myristylation, or both. In comparison, many other transmembrane proteinsdo not show a preference for lipid rafts; for example, CD45 and Ecadherin are excluded from these areas. Certain lipid modifiedtransmembrane proteins such as the HA molecule of influenza viruslocalize to lipid rafts.

Chemokine receptors (CRs), which serve as HIV co-receptors, areG-coupled proteins with seven membrane spanning domains, and belong tothe large family of serpentine receptors. The large number of membraneinteracting domains indicates that CRs can be more profoundly affectedby the lipids in the surrounding milieu than can a single passtransmembrane protein. For example, membrane cholesterol is essential inthe binding of the neuropeptide galanin to its G-coupled seven membranespanning receptor, GalR2. Precedence for cholesterol effects ontransmembrane protein function has been established by demonstratingthat cholesterol is required for ligand binding by two serpentinereceptors, the oxytocin receptor and the brain cholecystokinin receptor(Gimpl et al., Biochemistry 36:10959-10974, 1997), and the role ofcholesterol in receptor function has been attributed to association ofthe oxytocin receptor with lipid rafts (Gimpl and Fahrenholz, Eur. J.Biochem. 267:2483-2497, 2000). Similarly, as discussed above, theSemliki Forest virus (SFV) spike protein requires cholesterol andsphingolipids on target membranes for infection. Interestingly, thepresence of chemokine receptor 5 (CCR5) in lipid rafts on MCF7 cellscorrelates with its polarized distribution in chemotactic cells, but thefunctional correlation between CCR5 and lipid rafts has not been wellstudied. A role for lipid rafts in CXCR4 signaling has not beenestablished.

As disclosed herein, HIV-1 buds selectively from lipid rafts of infectedT cells (Example 1). In addition, to SFV, measles viruses, influenzaviruses, and polioviruses assemble by raft association and, in the caseof influenza virus, to bud from rafts (see, for example, Marquardt etal., J. Cell Biol. 123:57-65, 1993; Manie et al., J. Virol. 74:305-311,2000; Zhang et al., J. Virol. 74:4634-4644, 2000, each of which isincorporated herein by reference). The involvement of lipid rafts inHIV-1 biology beyond its role in virus budding has been furtherexamined. As further disclosed herein, partial depletion of cholesterolfrom cell membranes using a βCD inhibited HIV-1-induced syncytiumformation in cell lines and primary T cells (Example 2). βCD treatmentof cells also increased CR internalization induced by monoclonalantibody (MAb) binding. Primary cells and cell lines were renderedresistant to infection CXCR4-specific and CCR5-specific HIV-1 strains bytreatment with 2-OH-βCD (Example 2). The effects observed were not dueto loss of cell viability after treatment with the βCD, and demonstratethat intact lipid rafts and cholesterol are required for HIV-1 infectionand syncytium formation.

Since cholesterol is highly concentrated in lipid rafts and has beenimplicated in the entry of other viruses, the effect of lipid raftdispersion by cholesterol depletion on HIV-1 infection and syncytiumformation was examined. As disclosed herein, cholesterol is required forboth HIV-1 induced cell-cell fusion as well as infection by free virusparticles, similar to that reported for SFV, and contact of HIV-1 withβCD rendered the virus non-infectious. In the case of SFV, it appearsthat the cholesterol dependence can be attributed to the envelope spikeprotein. Another alphavirus, Sindbis virus, also requires cholesterol intarget membranes for infection (Lu et al., J. Virol. 73:4272-4278,1999). In vitro assays determined that cholesterol and sphingolipids arerequired in liposomes for fusion with Sindbis virus at low pH, even inthe absence of receptor (Smit et al., J. Virol. 73:8476-8484, 1999).Those studies established a clear requirement for cholesterol inmembrane fusion for the alpha viruses, and the present results indicatea similar role for cholesterol in HIV-1 fusion. The importance ofcholesterol for HIV-induced membrane fusion is also supported by studiesshowing that cholesterol in large unilamellar vesicles enhanced themembrane fusion activity of an HIV-1 gp41-derived peptide (Pereira etal., AIDS Res. Hum. Retrovir. 13:1203-1211, 1997).

Glycolipids are important components of lipid rafts and the role of hostglycolipids in HIV infection is being investigated. Inhibition ofsphingolipid synthesis by inhibitors such as PPMP(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol) reduces HIVinfection of CD4+ human cells by 50% (Puri et al., Biochem. Biophys.Res. Commun. 242:219-225, 1998). Moreover, CD4+ non-human cells weremade susceptible to gp120-gp41 mediated cell fusion by the addition ofhuman erythrocyte glycolipids (Puri et al., supra, 1998). CD4-inducedbinding of gp120 to glycosphingolipids Gb3 and GM3 from reconstitutedlipid raft microdomains also has been demonstrated (Hammache et al., J.Virol. 73:5244-5248, 1999). Those results suggest that glycolipids,which are enriched in lipid rafts, can also serve as cofactors indetermining viral tropism. Glycolipid-enriched membrane domains (lipidrafts) may serve as platforms for organizing CD4, CRs, and gp120/41 intofusion complexes (Hug et al., J. Virol. 74:6377-6385, 2000).

The results disclosed herein support a model for preferential HIV-1interactions with lipid rafts as sites for virus entry (Example 2). SV40also enters cells at lipid rafts (caveolae) even though its receptorappears to be MHC class I. The virus may bind to other regions of thecell membrane, but translocate to caveolae for entry. In addition,several bacterial toxins target lipid rafts as well. For example, thebacterial toxins aerolysin and Clostridium septicum alpha toxin bind toGPI-anchored proteins, which are highly enriched in lipid rafts, and theVibrio cholerae toxin binds to GM1. Cholera toxin oligomerization andpore formation in liposomes is promoted by cholesterol and sphingolipids(Zitzer et al., J. Biol. Chem. 274:1375-1380, 1999), and host derivedGPI-anchored proteins acquired by HIV-1 budding from lipid rafts rendersthe virus susceptible to neutralization by aerolysin (Nguyen et al.,Mol. Microbiol. 33:659-666, 1999).

The reduction of virus binding to cells treated with βCD likely involvesmore than βCD effects on CRs, since adhesion molecules also are involvedin virus binding to cells (Liao et al., AIDS Res. Hum. Retrovir.16:355-366, 2000, which is incorporated herein by reference). Theaffinities of adhesion molecules, including integrins LFA-1 and IVβ3,for their ligands are diminished by treatment of cells with βCD.Conversely, the addition of cholesterol increases binding of I5β1integrin to fibronectin, as well as increasing its localization to focaladhesions and interactions with the cytoskeleton. As disclosed herein,cholesterol depletion rendered CXCR4 sensitive to MAb-inducedinternalization not seen on control cells (Example 2). This resultindicates that cholesterol is involved in maintaining stable expressionof CXCR4. Furthermore, cells treated with βCD did not respond to SDF-1in LFA-1-mediated cell adhesion assays. This result demonstrates thatCXCR4, which normally regulates LFA-1 function, did not do so aftercholesterol depletion. Thus disruption of integrin function onβCD-treated cells can significantly diminish virus binding given thedemonstrated role of these molecules in HIV binding to cells.

Multiple extracellular loop domains of CXCR4 and CCR5 are believed to beinvolved in CR-gp120 binding and the subsequent conformational changesthat lead to HIV-1 fusion. Mouse CCR5 extracellular loop (ECL) loopswapping with human CCR5 revealed that all three loops are involved infunctional interaction with the HIV-1 envelope (Bieniasz et al., EMBO J.16:2599-2609, 1997). Env interactions with multiple ECLs of theco-receptor suggests that binding occurs in a groove or pocket at thelevel of the plasma membrane. Accordingly, a small molecule blockedgp120 interaction with CCR5 in a pocket formed between transmembranehelices 1, 2, 3, and 7. For CXCR4, antagonist peptide T22 blocked HIV-1infection by interacting with the N-terminus and at least ECL1 and ECL.Since CRs can project no further than a few nm above the plane of themembrane, gp120-CR interactions may bring their respective membranesinto close opposition to each other. Close membrane contact is requiredfor lipid intermixing between the two membranes after the triggering ofconformational changes in gp41. The requirement for conformationalintegrity of CR TM domains is evidenced by the finding that structuralanalogs of TM domains of CXCR4 and CCR5 inhibit signaling and HIVinfection. The insertion of these peptides is believed to disrupt theinteractions of the transmembrane helices in the CR, knocking out bothits ability to transmit signals and support HIV fusion. Thus changes inCR conformation in either their TM domains or ECLs can profoundly affecttheir ability to serve as HIV-1 co-receptors. The present resultsindicate that cholesterol is involved in maintaining functionalconformations of both CCR5 and CXCR4 (Example 2), a suggestion that issupported by results using the serpentine receptors, the oxytocin andcholecystokinin receptors (Gimpl et al., supra, 1997), where thereceptor function was strictly dependent on cholesterol, and from ligandbinding studies suggesting that depletion of cholesterol from the cellmembrane alters the conformation of these receptors.

Clustering of CXCR4 by cytoskeletal rearrangements can be important inHIV-1 cell entry and promoting chemotaxis of CD4 and CD8 cells (Hildrethand Orentas, Science 244:1075-1078, 1989, which is incorporated hereinby reference; see, also, Yang et al., J. Biol. Chem. 274:11328-11333,1999). Lipid raft aggregation induced by a chemotactic stimulationproduces similar cellular rearrangements (Manes et al., EMBO J.18:6211-6220, 1999). That redistribution of proteins, including CCR5 andthe T cell receptor, into lipid rafts appears is a critical trigger forcell function is supported by the finding that the removal ofcholesterol inhibits chemotaxis and cell polarization mediated throughCCR5 (Nieto et al., J. Exp. Med. 186:153-158, 1997; Lanzavecchia et al.,supra, 1999). Inhibition of HIV infection by cholesterol depletion mayreflect a similar requirement for these processes in HIV-1 infection.

Enhanced MAb-induced internalization of CR occurred after βCD depletionof cellular cholesterol (Example 2). Interestingly, the opposite effectwas observed in studies on the transferrin and epidermal growth factorreceptors, where βCD treatment reduced the rate of internalizationthrough clathrin coated pits. Previous studies on CXCR2 and CXCR4internalization induced by SDF-1I and PMA stimulation have shown thatthis process may be mediated by clathrin coated vesicles. Since βCDdepletion of cholesterol appears to inhibit coated vesicleinternalization, MAb-induced CR internalization in BCD-treated cells mayoccur through a distinct pathway, for example, similar to thedisplacement of caveolin from caveolae to the Golgi apparatus thatoccurs after cholesterol oxidase treatment of cells, which producesmembrane effects similar to cholesterol removal. Cholesterol depletionalso may alter CR interactions with other proteins at the cell membranethat are necessary for stable membrane expression.

Whether cholesterol is needed for conformational stability, stablemembrane expression, lipid raft mediated cell signaling, or all of theabove is not yet clear. Cholesterol removal does not strictly affectlipid rafts alone, but also can affect cell signaling and other cellularfunctions. However, the results on HIV-1 induced syncytium formation,which only requires expression of envelope protein and viralco-receptors at appropriate levels, indicate that intact lipid rafts andcholesterol play a critical role in the early steps of virus binding andfusion (Examples 1 and 2). These results extend previous reports showingfully reversible inhibition of HIV infection by depletion cholesterolfrom susceptible cells with βCD (Manes et al., EMBO J. 1: 190-196,2000). However, the latter studies were based primarily on transfectedepithelial cell lines (293, HeLa), and did not examine the effect of βCDtreatment on LFA-1, CD4 or CR expression, and in contrast to the presentresults, did not detect any reduction in HIV binding after βCDtreatment, perhaps because LFA-1-negative cells were used in the bindingassay.

HIV-1 prevention strategies must consider both cell-free andcell-associated virus because both HIV-1 virions and HIV-infected cellsare present in the semen and cervical mucus of infected individuals.Antibodies that target HIV-1 virions can prevent vaginal transmission ofcell-free virus in macaques. However, since cell-associated transmissionhas not been reliably demonstrated in these model systems, no strategiesto prevent such transmission have been tested. A model of vaginaltransmission using human peripheral blood leukocyte (HuPBL)reconstituted, severe combined immunodeficient (SCID) mice (HuPBL-SCIDmice), in which cell-associated HIV-1 transmission occurs and ismediated by transepithelial migration of HIV-infected cells, isdescribed (Khanna et al., 2001), and was used to demonstrate thattopically applied βCD blocks transmission of cell-associated HIV-1(Example 3). These results also demonstrate that the HuPBL-SCID model ofvaginal HIV-1 transmission is useful for investigating cell-associatedtransmucosal HIV-1 transmission, and for screening reagents for theirpotential efficacy in preventing sexual transmission of pathogens suchas HIV. The HuPBL-SCID mouse model provides a means to screen largenumbers of animals to determine the statistical robustness ofobservations made using a pathogen of interest. Thus, while the utilityof the model is exemplified by addressing the role of cell-associatedtransmission of HIV-1, it will be recognized that the model also isuseful for examining other sexually transmitted pathogens that sharefeatures of HIV-1 transmission clinically, including, for example, thetransmission of viruses that use CCR5 as a co-receptor.

The results demonstrating that HIV-1 transmission to vaginal cells bytreatment with βCD were extended to another sexually transmittedenveloped virus, HSV-2. As disclosed herein, contact of HSV-2 virus with2-OH-βCD significantly reduced vaginal infectiousness of the virus in amouse genital herpes model system (see Example 4; see, also, Sherwood etal., Nature Biotechnol. 14:468-471, 1996; which is incorporated hereinby reference). These results demonstrate that βCD is useful for reducingthe risk of transmission of a variety of sexually transmitted pathogens,including sexually transmitted enveloped viruses.

The migration of HIV-infected cells and the movement of assembled virusparticles out of infected donor cells are critical to HIV-1transmission. As disclosed herein, HIV-1 budding occurred selectivelythrough lipid rafts on the cell surface (Examples 1 and 2). In addition,the ability of lipid rafts to act as adhesion platforms facilitatescell-cell interactions and migration, which may be important forcell-to-cell transfer of virus and for entry of infected cells throughgenital tract epithelia, respectively (Krauss and Altevogt, J. Biol.Chem. 274:36921-36927, 1999; Manes et al., supra, 1999). βCDs, which arewater soluble compounds that disrupt lipid rafts by removing cholesterolfrom cellular membranes, interrupt cellular migration (Okada et al., J.Pharm Exp. Ther. 273:948-954, 1995) and inhibit syncytium formation ofHIV-1 infected cells (see Example 2).

The HuPBL-SCID mouse model was used to examine the ability of the βCDderivative, 2-OH-βCD, to interrupt cell-associated transmission ofHIV-1. As disclosed herein, intravaginal administration of a βCD priorto challenge by HIV-1 infected cells efficiently blocked virustransmission and induced minimal, if any, damage to the vaginal mucosa(Example 3). In addition, a mouse genital herpes model system was usedto demonstrate that βCD treatment can reduce the risk of transmission ofcell-free HSV-2 (Example 4). These results demonstrate that animalmodels for vaginal transmission of sexually transmitted diseases can beused to screen βCD derivatives in a cost-effective way, thus providing ameans to identify βCDs that can reduce the risk of transmission ofsexually transmitted pathogens and that do not cause undue toxicity tonormal healthy tissue.

Several mechanisms have been proposed by which HIV-1 is able to traversethe epithelium of the genitourinary tract to establish productiveinfection in lymph nodes. For example, HIV-1 can be transmitted frominfected lymphocytes to epithelial cells, or through the epithelium,which serves as a conduit through which virus is transcytosed,presumably to cells within the lamina propria that are susceptible toproductive infection. Intravaginal inoculation of rhesus macaques withSIV demonstrated rapid association of the virus with dendritic cellsadjacent to or between the epithelial cells lining the genitourinarytract (Miller and Hu, J. Infect. Dis. 179(Suppl. 3):S413-417, 1999), orto quiescent T cells similarly placed in the reproductive tract (Zhanget al., Science 286:1353-1357, 1999). All of these mechanisms oftransmission involve exposure of free virus to the extracellularenvironment, providing an opportunity, albeit a brief one, for virusspecific intervention strategies to be effective at the mucosal surface.Of additional concern, however, is the possibility that lymphocytes ormacrophages from the infected donor could migrate directly through theepithelium of the genitourinary tract to infect lymphocytes in lymphnodes draining the genitourinary tract. As such, anti-HIV antibodies orother virion-specific strategies, while important and perhaps necessaryfor a protective effect, may not be sufficient to prevent transmissionof the virus.

Migrating cells carrying HIV have been referred to “Trojan horse”leukocytes because of their ability to hide the virus fromvirus-specific defenses that may be present within the genitourinarytract (Anderson and Yuni, New Engl. J. Med. 309:984-985, 1983). Whileconsiderable effort has been directed to identifying virus-specificintervention strategies effective against sexual transmission of humanand simian immunodeficiency viruses, there has been little effort toidentify strategies for interrupting migration of infected cells toregional lymph nodes. Use of a mouse model of vaginal transmissiondemonstrated vaginal transmission of HIV-1 using infected-cell inocula(Example 3). The HuPBL-SCID model is unique in that the processes ofcell-associated HIV-1 transmission can be examined in the absence of thepossibility of that cell-free virus is mediating transmission. In fact,the amount of infectious virus produced by the number of infected cellsin the inocula used in the present study would be predicted to bedramatically less than 1×10⁶ TCID₅₀ of free virus that failed to infect(Burkhard et al., AIDS Res. Hum. Retrovir. 13:347-355, 1997).

In the HuPBL-SCID mouse system, HIV-1-infected PBMC can migrate throughcervix-like epithelium to regional lymph nodes (Example 3). As such, themice can be used to evaluate strategies for effectively blockingcell-associated HIV-1 transmission. To date, cell-associated SIV has notbeen successfully transmitted by the vaginal route in a macaque model,although both cell-free and cell-associated HIV-1 have been transmittedby viral inoculations at the cervical os of chimpanzees. Similarly, bothcell-free and cell-associated feline immunodeficiency virus have beentransmitted in cat models of vaginal infection.

In the HuPBL-SCID mice, only CCR5-utilizing HIV-1 can be transmitted andestablish infection in the HuPBMC that were transplantedintraperitoneally into the mice. It is unclear whether this preferentialtransmission reflects preferential movement of CCR5-utilizingvirus-infected cells across the mucosal barrier, or an enhanced abilityof these viruses to continue productive infection in the unactivatedHuPBMC residing in the peritoneal cavity seven days after human celltransplantation. Nevertheless, this finding parallels the observationthat viruses that can use CCR5 as a co-receptor for entry arepreferentially transmitted in the clinical setting.

Unlike other model systems of vaginal transmission, the HuPBL-SCID mousemodel of transmission is dependent upon the movement of virus-infectedcells to sites at which other human cells exist, in this case theperitoneal cavity of the infected mice. Human cells transplanted intothe peritoneum do not appear to migrate to the vaginal mucosa orsub-mucosa. As such, the inability of free virus to be transmitted inthis system may simply reflect a poor migratory ability of free virusand the absence of human target cells within and beneath the vaginalmucosa. Thus, the results disclosed herein do not indicate that freevirus is not transmitted in the clinical setting but, instead,demonstrate that infected-cell migration through cervical epitheliummust be considered in any intervention strategy.

The migration of mononuclear cells through murine vaginal epithelium hasbeen documented (see, for example, Ibata et al., Biol. Reprod.56:537-543, 1997; Zacharapoulos et al., Curr. Biol. 7:534-537, 1997).The results disclosed herein reinforce the notion that the single layerof columnar epithelial cells present on the surface of the cervix ismore susceptible to transmigration of HIV-infected PBMC and, conversely,that the stratified squamous epithelium lining the normal vagina is lessvulnerable to transepithelial transmission, presumably by reducing theefficacy of transepithelial migration. Progesterone treatment of themice effectively converted the vaginal stratified squamous epitheliuminto a cervix-like columnar epithelium, thus greatly increasing thesurface area within the vagina that is covered with columnar epithelium.

The HuPBL-SCID model of vaginal transmission has allowed confirmationthat a βCD derivative is highly effective at interrupting vaginaltransmission of cell-associated HIV-1. Application of the βCD to thevaginal mucosa prior to inoculation with HIV-1 infected cellsdramatically reduced transmission of cell-associated virus (Example 3).βCDs are cyclic, water-soluble carbohydrates that are comprised of sevenglucose units and have been used clinically as a food additive (Toyodaet al., Food Chem. Toxicol. 35:331-336, 1997) and as a molecularcomplexing agent that can increase the solubility and stability of somepoorly soluble drugs (Sharma et al., J. Pharm. Sci. 84:1223-1230, 1995).As disclosed herein, βCD applied to the vaginal mucosa was substantiallyless toxic than a sub-clinical concentration of the widely usedspermicide nonoxynol-9 (see Example 3).

Migration through the epithelium likely involves, as an initial step,interaction between lymphocytes and/or macrophages and epithelial cells.Clustering of lipid rafts on cell membranes results in enhancement ofcell-cell interactions and migration, and disruption of the rafts withβCD diminishes cell binding and migration. Moreover, the production ofHIV-1 virions from such cholesterol-depleted cells is dramaticallydecreased and these virions are significantly less infectious. Theresults disclosed herein demonstrate that the HuPBL-SCID mice of vaginaltransmission of cell-associated virus provides a simple and inexpensivesystem to identify agents that can be used in vaginal products forpreventing sexual transmission of HIV-1 (Example 3).

As disclosed herein, 2-OH-βCD significantly blocked vaginal transmissionof cell-associated HIV-1 and of cell-free transmission of HSV-2(Examples 3 and 4). Since this agent is currently used for humanadministration, it will be recognized that 2-OH-βCD can be used alone,or in combination with other agents such as a contraceptive orantibiotic, to reduce the risk of transmission of sexually transmitteddiseases. Accordingly, the present invention also provides methods forreducing the risk of transmission of a sexually transmitted pathogen toor from a subject. As such, a method of the invention can be performedwith respect to the infected individual, thus reducing the risk that thesubject will transmit the disease to an uninfected subject, or can beperformed with respect to an uninfected individual, thus protecting thesubject from an infected individual, who may or may not know he or sheis infected. Where the method is used to prevent transmission from aninfected individual to an uninfected individual, the pathogen or cellssusceptible to infection by the pathogen can be contacted with the βCDin the infected subject, in the uninfected subject, or in both. Thesubject can be any subject susceptible to a sexually transmitteddisease, and generally is a vertebrate subject, particularly a mammal,and preferably a human.

A method of the invention can be performed, for example, by contactingthe sexually transmitted pathogen or cells susceptible to infection bythe pathogen in an uninfected subject with a pharmaceutical compositioncomprising a βCD, thereby reducing the risk of the subject becominginfected with the sexually transmitted the pathogen. It should berecognized that a method of the invention can reduce the risk oftransmission of various sexually transmitted diseases. As such, evenwhere a subject already is infected with one or more sexuallytransmitted pathogens, a method of the invention can reduce the risk ofinfection by other sexually transmitted pathogens. A method also can beperformed, for example, by contacting the pathogen or the cellssusceptible to infection by the pathogen in a subject having a sexuallytransmitted disease with a pharmaceutical composition comprising a βCD,thereby reducing the risk of transmission of the sexually transmitteddisease by the subject to another individual.

The present invention also provides compositions useful for reducing therisk of transmission of sexually transmitted disease. A composition ofthe invention contains a βCD, which can be in a form suitable fortopical administration to a subject, particularly intravaginal orintrarectal use, including a suppository, a bioadhesive polymer, or avaginal disk, which can provide timed release of the βCD (see, forexample, U.S. Pat. Nos. 5,958,461 and 5,667,492, each of which isincorporated herein by reference; see, also, Sherwood et al., supra,1996); or can be formulated in combination with a solid substrate toproduce a condom, diaphragm, sponge, tampon, a glove or the like (see,for example, U.S. Pat. Nos. 6,182,661 and 6,175,962, each of which isincorporated herein by reference), which can be composed, for example,of an organic polymer such as polyvinyl chloride, latex, polyurethane,polyacrylate, polyester, polyethylene terephthalate,poly(ethylene-co-vinyl acetate); polymethacrylate, silicone rubber, asilicon elastomer, polystyrene, polycarbonate, a polysulfone, or thelike (see, for example, U.S. Pat. No. 6,183,764, which is incorporatedherein by reference; see, also, Sherwood et al., supra, 1996).

For topical administration, the βCD can be formulated in anypharmaceutically acceptable carrier, provided that the carrier does notaffect the activity of the βCD in an undesirable manner. Thus, thecomposition can be, for example, in the form of a cream, a foam, ajelly, a lotion, an ointment, a solution, a spray, or a gel (see U.S.Pat. No. 5,958,461, which is incorporated herein by reference). Inaddition, the composition can contain one or more additional agents, forexample, an antimicrobial agent such as an antibiotic or anantimicrobial dye such as methylene blue or gentian violet (U.S. Pat.No. 6,183,764); an antiviral agent such as a nucleoside analog (e.g.,azacytidine), a zinc salt (see U.S. Pat. No. 5,980,477, which isincorporated herein by reference), or a cellulose phthalate such ascellulose acetate phthalate or a hydroxypropyl methylcellulose phthalate(see U.S. Pat. No. 5,985,313, which is incorporated herein byreference); a contraceptive (see U.S. Pat. No. 5,778,886, which isincorporated herein by reference); a lubricant, or any agent generallyuseful to a sexually active individual, provided the additional agent,either alone or in combination, does not affect the activity of the βCDor, if it affects the activity of the βCD, does so in a predictable waysuch that an amount of βCD that is effective for reducing the risk oftransmission of a sexually transmitted pathogen can be determined.

A pharmaceutically acceptable carrier useful in a composition of theinvention can be aqueous or non-aqueous, for example alcoholic oroleaginous, or a mixture thereof, and can contain a surfactant,emollient, lubricant, stabilizer, dye, perfume, preservative, acid orbase for adjustment of pH, a solvent, emulsifier, gelling agent,moisturizer, stabilizer, wetting agent, time release agent, humectant,or other component commonly included in a particular form ofpharmaceutical composition. Pharmaceutically acceptable carriers arewell known in the art and include, for example, aqueous solutions suchas water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters. A pharmaceutically acceptable carrier can containphysiologically acceptable compounds that act, for example, to stabilizeor to increase the absorption of the βCD, for example, carbohydrates,such as glucose, sucrose or dextrans, antioxidants, such as ascorbicacid or glutathione, chelating agents, low molecular ++weight proteinsor other stabilizers or excipients.

The pharmaceutical composition also can comprise an admixture with anorganic or inorganic carrier or excipient suitable for intravaginal orintrarectal administration, and can be compounded, for example, with theusual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, orother form suitable for use. The carriers, in addition to thosedisclosed above, can include glucose, lactose, mannose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695).

The βCD also can be incorporated within an encapsulating material suchas into an oil-in-water emulsion, a microemulsion, micelle, mixedmicelle, liposome, microsphere or other polymer matrix (see, forexample, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. 1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981),each of which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer. “Stealth” liposomes (see U.S.Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each of which isincorporated herein by reference) are an example of such encapsulatingmaterials particularly useful for preparing a pharmaceutical compositionof the invention, and other “masked” liposomes similarly can be used,such liposomes extending the time that the βCD remains at the site ofadministration.

The composition generally is used at or about the time of sexualactivity, and usually is used prior to initiating sexual contact. Themanner of use will depend, in part, on the form of the composition, forexample, whether the composition is in a liquid or liquid-like form suchas a jelly, a douche, a cream or the like, or whether the βCD isformulated with a solid substrate such as a sponge, diaphragm, tampon,pessary, condom or the like. When formulated as such a composition, theβCD can be impregnated into an absorptive material such as a sponge ortampon, or coated onto the surface of a relatively impermeable solidsubstrate such as a condom or diaphragm, or on medical gloves, thusproviding a means to contact the βCD with the pathogen or cells in asubject that are susceptible to infection.

The amount a βCD in a composition can be varied, depending on the typeof composition, such that the amount present is sufficient to reduce theability of the pathogen to be sexually transmitted. An effective amountof a βCD can block infection of susceptible cells by a sexuallytransmitted pathogen such as free HIV, or cell-associated HIV present ina secretion, or by uptake of the pathogen due to binding to otherwisenon-susceptible cells, which then transfer the sexually transmittedpathogen to susceptible cells. An example of such an amount is about 1to 100 mM, generally about 5 to 30 mM, when administered in an ointment,gel, foam, spray or the like, our about 0.1 to 2 grams, generally about0.25 to 0.75 grams, when administered as a suppository or in combinationwith a solid substrate. An effective amount of a βCD also can bemeasured in a weight:weight (w:w) or weight:volume (w:v) amount, forexample, about 0.1% to 3% w:w with respect to a solid substrate or about0.1% to 3% w:v with respect to a pharmaceutically acceptable carrier. Inaddition, an amount of a βCD sufficient to reducing the risk oftransmission of a sexually transmitted disease can be determined usingroutine clinical methods, including Phase I, II and III clinical trials.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 HIV-1 Selectively Buds From Lipid Rafts

This example demonstrates that HIV-1 budding occurs through lipid rafts,thereby accounting for the cholesterol-rich, sphingolipid-rich virusmembrane, which bears GPI-linked proteins such as Thy-1 and CD59, butlacks CD45.

Cells and Antibodies

Jurkat cells were obtained from the American Type Culture Collection(Rockville Md.) and maintained in complete medium, cRPMI, consisting ofRPMI 1640 (Gibco BRL/Life Technologies; Gaithersburg Md.) containing 10%fetal calf serum (FCS; HyClone; Logan Utah) and 10 mM HEPES. Monoclonalantibodies (MAbs) to Thy-1 (5E10) and CD59 (p282/H19) were obtained fromPharmingen (San Diego Calif.). Mouse MAb against HIV p17 was obtainedfrom Advanced Biotechnologies, Inc. (Columbia Md.). Goat anti-choleratoxin B (CTB) MAb was purchased from Calbiochem (La Jolla Calif.).Rabbit anti-GM1 polyclonal antibody was purchased from Metraya, Inc.(Pleasant Gap Pa.). Biotinylated human anti-HIV polyclonal antibodieswere produced from pooled human HIV 1 sera. Soluble recombinantCD4-immunoglobulin Fc chimera (CD4Ig) was obtained from (Genentech;South San Francisco Calif.). Control mouse myeloma immunoglobulin G1(IgG1) and rabbit anti-mouse IgG (Fc specific) were purchased fromJackson Immunoresearch (West Grove Pa.). Fluorescein isothiocyanate(FITC)-conjugated sheep anti-human IgG was purchased from CappelResearch Products (Durham N.C.). MAbs to major histocompatibilitycomplex I (MHCI) antigen (MHM.5), HIV-1 Gag (Gag.M1), and CD45 (H5A5)were produced as previously described, and were purified from ascitesfluids (Ellis et al., Hum. Immunol. 13:13-19, 1985; Hildreth and August,J. Immunol. 134:3272-3280, 1985, each of which is incorporated herein byreference).

Virus Production

HIV-1_(RF) was used to chronically infect Jurkat cells. Viruses used forthe capture assay were produced by washing 1×10⁶ to 2×10⁶ chronicallyinfected cells with phosphate-buffered saline (PBS), resuspending cellsin complete medium, and culturing for 1 to 3 days before collectingculture supernatants. Virus production was measured by p24 enzyme-linkedimmunosorbent assay (ELISA) after detergent lysis of supernatant.

Flow Cytometry

Flow cytometry was performed as previously described (Orentas andHildreth, supra, 1993). Briefly, 2×10⁵ cells in 100 ml of PBS containing5% normal goat serum (NGS) were added to 100 ml of MAb (1 to 5 mg) andincubated for 30 min on ice. Cells were washed with PBS, resuspended in100 ml of PBS plus 5% NGS containing 2 mg of FITC-goat anti-mouse IgG(FITC-GAM), and incubated 1 hr on ice. Cells were then washed with PBSand fixed with 2% paraformaldehyde, followed by analysis on an EPICSProfile II (Coulter; Hialeah Fla.) flow cytometer.

Virus Phenotyping

Virus phenotyping was carried out as previously described with someminor differences (Orentas and Hildreth, supra, 1993). Briefly, CostarELISA plates (Costar; Cambridge Mass.) were coated for 4 hr at 37° Cwith 1.5 mg of rabbit anti-mouse IgG (Fc fragment specific) per well in50 mM Tris (pH 9.5). The wells were blocked with 3% bovine serum albumin(BSA) in PBS for 2 hr at 37° C. before adding 1 to 2 mg of the MAbs. Theplates were then incubated overnight at room temperature before washingthem six times with PBS-0.05% Tween 20. Viral supernatants werecollected and clarified through 0.45 Tm (pore-size) filters. The viralsupernatants at 466 ng/ml of p24 were added to the antibody-coated wellsand incubated at 37° C. for 1.5 hr before washing them six times withRPMI. The bound viruses were then lysed with 1% Triton X-100 in cRPMIfor 1 hr at 37° C. Detergent-solubilized viral proteins were transferredto a second plate to measure released p24 in a standard p24 ELISA.

Cell Capture Assay

Costar ELISA plates were coated overnight at room temperature with 1.0mg of GAM IgG (Fc specific) per well in 50 mM Tris (pH 9.5). Wells wereblocked with 3% BSA in PBS for 1 hr at 37° C. before adding 1 to 2 mg ofthe MAbs. Plates were then incubated for 2 hr at 37° C. before washingthem three times with RPMI. Wells were blocked again with 5% NGS in PBSfor 1 hr at 37° C. before washing them three times with RPMI. Jurkatcells (10⁷) were labeled with horseradish peroxidase (HRP; Sigma) at 1mg/ml in cRPMI for 30 min at 37° C., washed once with cRPMI, thenresuspended in cRPMI to make 2.5×10⁶ cells/ml. Cells (100 ml) were addedto the wells and allowed to settle for 2 hr at 37° C. Wells were washedthree times with Hanks balanced salt solution (Gibco BRL), then treatedwith lysis-substrate buffer (1% Triton X-100, 0.015% H₂O₂, 0.24 mg oftetramethylbenzidine per ml, 0.2M sodium acetate-citric acid; pH 4.0)for 20 min before the addition of 0.5 M H₂SO₄ to stop the reaction.Absorbances at a 450-nm wavelength were determined on a plate reader,and cell number values were extrapolated from a linear curve.

β-Cyclodextrin Treatment and Virus Precipitation

Infected Jurkat cells (3×10⁶) were treated with 20 mMhydroxypropyl-β-cyclodextrin (2-OH-βCD; Cyclodextrin TechnologiesDevelopment, Inc.; Gainesville Fla.) in 3 ml of cRPMI or with cRPMIalone for 1 hr at 37° C. Cells were washed with PBS, then allowed toproduce virus in 3 ml of cRPMI at 37° C. for 2 hr. Viral supernatantswere clarified through a 0.45 Tm filter, and 100 ml was added to 100 mlof MAb (10 mg/ml) in 5% NGS-PBS, and the mixture was incubated for 1 hron ice. Pansorbin (SaC) (50 mg; Calbiochem; San Diego Calif.) was addedto the solution and incubated for 20 min on ice. Complexes were washedsequentially with 10X and 1X PBS. Precipitated virus was lysed with 400ml of 1% Triton X-100 in cRPMI. Lysates were diluted, and p24 wasquantitated by standard p24 ELISA.

Cholera Toxin Capture of HIV-1

HIV-1_(RF) viral supernatant from an infected Jurkat cell line wascollected and clarified through a 0.45 Tm filter. Virus supernatant (100ml) was added to 100 ml of CTB (Calbiochem) dilutions (0 to 20 mg/ml) incRPMI. The mixtures were incubated for 1 hr at 37° C. before adding 50ml of goat anti-CTB at 10 mg/ml in 5% NGS-PBS. The mixture was thenincubated for 1 hr on ice before adding 50 ml of SaC, mixed well, thenincubated on ice for another 1 hr with intermittent mixing. The SaC waswashed twice with PBS, and SaC-precipitated virus was lysed with 400 mlof 1% Triton X-100 in cRPMI at room temperature for 30 min. Released p24was measured with a standard p24 ELISA after pelleting the SaC.

Immunomicroscopy

Cell surface staining of chronically infected cells and uninfected cellswas performed under saturating conditions. Jurkat cells (3×10⁵) werewashed in cold PBS and preincubated on ice for 15 min in 5% NGS-PBS.Uninfected cells were incubated with 1 to 5 mg of MAb in 5% NGS-PBS for30 min on ice, washed with PBS, and incubated with 2 mg of Texasred-conjugated GAM IgG. Infected cells were incubated with biotinylatedhuman anti-HIV polyclonal antibody (10 mg/ml in 5% NGS-PBS) for 30 minon ice and washed with PBS before incubating them with 2 mg of Texasred-streptavidin conjugate. Both cell types were incubated with thesecond primary MAb at 1 to 5 mg in 5% NGS-PBS 30 min on ice, washed withPBS, and incubated with 2 mg of FITC-GAM in 5% NGS-PBS. The cells werethen fixed with 2% paraformaldehyde in PBS and cytospun ontopoly-L-lysine-coated slides by using Cyto Funnels (Shandon; PittsburghPa.). The pellets were overlaid with 50 ml of 25% glycerol in PBS, and acoverslip was positioned over the droplet. The edges of the slides weresealed with nail polish before storing them at 4° C. This stainingprocedure was also performed with cells prefixed with 2%paraformaldehyde in PBS prior to MAb staining. Viewing of slides wasper-formed with an Olympus IX50 confocal microscope under oil immersionat an ×100 magnification. Micrographs were analyzed on a SiliconGraphics Work-station with Intervision software. Final images wereenhanced on the Silicon Graphics Workstation by two-dimensionaldeconvolution, and brightness and contrast were adjusted for viewing.

Cell Lysis and Equilibrium Centrifugation

Protein extraction and equilibrium centrifugation were performed aspreviously described with slight modifications (Ilangumaran et al.,Anal. Biochem. 235:49-56, 1996, which is incorporated herein byreference). Briefly, 2×10⁷ cells were washed twice in PBS and once inTKM buffer (50 mM Tris-HCl, pH 7.4; 25 mM KCl; 5 mM MgCl₂; 1 mM EDTA).Cells were extracted on ice for 30 min in 500 ml of lysis buffer (TKM,1% Triton X-100, 2 mg of aprotinin per ml). Lysates were centrifuged at8,000×g for 10 min at 4° C., and the supernatants were stored at ⁻20° C.For equilibrium centrifugation, extracts were adjusted to 40% sucrose inTKM and loaded into SW41 tubes. The extracts were overlaid with 6 ml of38% sucrose-TKM, followed by 4.5 ml of 5% sucrose-TKM. Tubes werecentrifuged at 100,000×g for 18 hr at 4° C. Eleven 1 ml fractions werecollected from the bottom of the tube and stored at ⁻20° C.

Dot Immunoassay

Dot immunoassays were performed as described previously with minormodifications (Ilangumaran et al., supra, 1996). Briefly, 100 mlportions of each fraction diluted 1:10 in PBS (2×10⁵ cell equivalents)were added to wells of a Bio-Dot apparatus (Bio-Rad; Hercules Calif.),gently suctioned onto nitrocellulose membranes, and allowed to air dry.The membranes were cut into strips and stored at ⁻20° C. in plasticbags. Before blotting, strips were blocked with 5% nonfat milk powder inTBST (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 0.1% Tween 20) for 1 hr atroom temperature. Strips were then incubated with primary antibodies inTBST/0.5% milk powder for 1 hr, and washed 10 min three times with TBST,followed by incubation with HRP-conjugated GAM for 45 min. The stripswere then washed five times and developed with an enhancedchemiluminescence (ECL) assay (Amersham Life Science; Arlington HeightsIll.) before exposure to Hyper-Film ECL.

Dialkylindocarbocyanine Labeling

Three million Jurkat HIV-1_(RF)-infected cells were washed with PBS andresuspended in 1 ml of cRPMI. DiIC16 or DiIC12(1,19-didodecyl-3,3,39,39-tetramethylindocarbocyanine; Molecular Probes;Eugene Oreg.; Arthur et al., supra, 1992) in 0.1 mg of ethanol per mlwas added to make a final concentration of 1 to 10 mM. Cells wereincubated on ice for 15 min to allow the incorporation of dyes. Thecells were washed with PBS and fixed with 2% paraformaldehyde in PBSbefore further MAb labeling for confocal microscopy.

(³H)-Myristic Acid Labeling and Immunoprecipitation

HIV-1-infected Jurkat cells (2×10⁷) were labeled in 2 ml of cRPMIcontaining 1 mCi of (9,10(n)-³H)-myristic acid (40 to 60 Ci/mmol;Amersham Pharmacia Biotech.; Piscataway N.J.) for 4 hr at roomtemperature. Labeled cells were lysed and subjected to sucrose gradientequilibrium centrifugation as described above. GEM domain (lipid raft)fractions 3, 4, and 5 were pooled as were soluble fractions 8, 9, and10. Samples were pre-cleared by incubation with 20 ml of normal humanserum for 1 hr at 4° C. before adding 100 ml of SaC and incubating theman additional 30 min. The preimmune complexes were removed, and sampleswere incubated with excess IgG1 myeloma or Gag.M1 MAb for 1.5 hr at 4°C., followed by the addition of 2 mg of RAM (Fc specific). After 1 hr,50 ml of SaC was added, followed by incubation for 30 min. Immunecomplexes were washed twice with PBS and resuspended in 200 ml of PBS.Samples were then boiled, and the supernatant was blotted onto anitrocellulose membrane with a Bio-Dot apparatus. The membrane wastreated with En³Hance Spray (DuPont; Wilmington Del.), then exposed toHyperfilm-MP (Amersham) for 5 days. Dots were quantitated bydensitometry analysis by MacBAS software version 2.5, and the percentdistribution in GEM domains was determined by using the followingformula:(Gag_(GEM−IgG) _(GEM))/{(Gag_(GEM)−IgG_(GEM))+(Gag_(So)l−IgG_(Sol))}.Results

Microfluorimetry of infected Jurkat cells showed high expression of CD45and low expression of Thy-1 and CD59. Flow cytometry under saturatingconditions was used to determine the expression of CD45, Thy-1, and CD59on the surface of infected Jurkat cells. CD45 was highly expressed onJurkat cells (see Nguyen and Hildreth, J. Virol. 74:3264-3272, 2000,which is incorporated herein by reference; see FIG. 1). An antibodyagainst MHCI was used as a positive control, while mouse myelomaimmunoglobulin (IgG1) was used as a negative control. Expression ofThy-1 and CD59 were significantly lower than that of CD45 and MHCI.These results correlate with previous surface expression analyses(Orentas and Hildreth, supra, 1993) and was corroborated by conventionalimmunofluorescence staining.

HIV-1 incorporated the GPI-linked proteins, Thy-1 and CD59, andganglioside GM1. The virus phenotyping assay, in which HIV-1 particlesare captured by MAbs through host proteins present on the viral particlesurface, was used to determine the relative host protein phenotype ofHIV-1 particles. The relative p24 captured by the MAbs was determined inthree experiments. MAbs to gp41 and MHC I captured virus efficiently.Thy-1 and CD59 also supported efficient viral capture despite lowexpression on the host cell surface. However, very little HIV-1 wascaptured through CD45 despite very high expression of CD45 on the cellsurface. The failure of the anti-CD45 MAb (H5A5) to capture HIV-1 wasnot due to low MAb affinity or failure to bind to the capture plate (seeOrentas and Hildreth, supra, 1993). The H5A5 MAb also was capable ofcapturing HRP-labeled Jurkat cells in a similar assay as efficiently asMAbs against other membrane proteins (see Nguyen and Hildreth, supra,2000; FIG. 1). Thus, the failure of anti-CD45 MAb to capture virus wasnot due to a failure of the MAb to work in the capture assays.

These results demonstrate a significant preference for HIV-1incorporation of GPI-linked proteins as compared to CD45. The highexpression of CD45 on the cell surface and its low incorporation intovirus particles was consistent with exclusion of this molecule frombudding particles. To corroborate the MAb plate capture assay results,HIV-1 immunoprecipitations were performed with MAbs. This assay allowedfor the potential interaction of all the virions in solution with theMAbs, in contrast to the plate virus capture assay, in which only asmall fraction of the particles make contact with the MAbs. Theanti-gp41 MAb, T32, which was used as a positive control for intactvirions, precipitated up to 60% of the p24 in the supernatant, dependingon the virus preparation. The anti-CD59 MAb precipitated as much p24 asanti-gp41 MAb T32. However, even in this assay, anti-CD45 MAb capturedvery little virus.

The effects of 2-OH-βCD, a cellular cholesterol efflux inducingmolecule, on the incorporation of host molecules into virions also wasexamined. By removing cholesterol, 2-OH-βCD is believed to partiallyperturb organized lipid rafts, resulting in dispersal of theircomponents (Ilangumaran and Hoessli, Biochem. J. 335:433-440, 1998). Thecapture of HIV-1 by MAbs against CD59 and gp41 decreased substantially(P<0.05) after treating cells with 2-OH-βCD, as measured by thepercentage of total p24 (see Nguyen and Hildreth, supra, 2000; FIG. 2).The decrease in Thy-1 was not statistically significant (P=0.08). CD45capture remained mostly unaffected. The effects on virus precipitationthrough gp41 indicate that intact lipid rafts are required for efficientgp41 incorporation into virions, since the overall cellular release ofp24 actually increased after 2-OH-βCD treatment.

The relative incorporation of GM1, a ganglioside marker specific forlipid rafts, also was examined. Using a soluble CTB binding assay, asmuch as 75% of HIV-1 was precipitated using goat anti-CTB and SaC aftertreating the virus with GM1-specific CTB (see Nguyen and Hildreth,supra, 2000; FIG. 3). The CTB binding to virus was specific and dosedependent, and no virus was precipitated in the absence of CTB asmeasured by p24 ELISA. These results demonstrate that the majority ofHIV-1 particles incorporated the lipid raft-specific marker GM1.

Thy-1, CD59, and GM1 colocalized with HIV-1 proteins on infected celluropods, which excluded CD45. To determine the distribution of HIV-1proteins relative to GPI-linked proteins that serve as lipid raftmarkers, infected cells were subjected to immunofluorescence stainingfollowed by confocal microscopy. Expression of HIV-1 proteins waslocalized to uropods projecting from one end of the cell. This cappingpattern was seen on most cells in the infected cell culture. Uropodsprotruding from HIV-1-infected cells have been described for adherent Tcells. Thy-1 and CD59 both colocalized with cell surface HIV-1 proteins,as shown by a superimposed green (Thy-1 or CD59) and red (HIV-1proteins) fluorescence (see Nguyen and Hildreth, supra, 2000; FIG. 4).Cells that were prefixed with 2% paraformaldehyde before staining showeda similar appearance, indicating that the colocalization was not due toantibody crosslinking of viral and GPI-linked proteins. Since the cellswere not permeabilized before staining, the HIV proteins seen in thesestudies are likely gp41 and gp120. This was confirmed in studies withanti-gp41 MAb T32 in the colocalization studies. Uninfected cells showedno capping of Thy-1 or CD59. CD45 did not localize to areas of HIV-1protein expression and was excluded from uropods. The distribution ofCD45 was unaffected by HIV-1 infection, and the molecule remained evenlydispersed in patches all over the cell surface. These results confirmthose obtained using the virus phenotyping studies. The ability of GM1to colocalize on the cell surface with HIV-1 proteins was examined toconfirm the finding that GM1 was present on virions. GM1 staining wasrelatively faint with rabbit anti-GM1 antibody, but confocal microscopyshowed colocalization of this molecule with HIV-1 labeled cells.

The lipid raft-partitioning lipid analog, DiIC16, colocalizes with HIV-1proteins on uropods of infected cells. In order to evaluate thelocalization of lipids in lipid rafts, two forms ofdialkylindocarbocyanine, a fluorescent lipid analog, were used—DiIC16,which partitions preferentially to lipid-ordered domains due to its two16-carbon saturated fatty acid chains, and DiIC12, which, with its two12-carbon saturated fatty acid chains, partitions to fluid domains.Infected Jurkat cells were labeled with the dyes for 15 min on ice,washed with PBS, and fixed with 2% paraformaldehyde in PBS. Cells werethen stained with soluble CD4Ig or human anti-HIV polyclonal antibodyand FITC-labeled sheep anti-human IgG to detect surface gp120/41.

Confocal microscopy showed that cells labeled with DiIC16 extensivelycolocalized with HIV-1 proteins (see Nguyen and Hildreth, supra, 2000;see FIG. 5). In contrast, DiIC12 did not preferentially label uropodsand was specifically excluded from areas with HIV-1 staining. Asexpected, CD45 staining was also excluded from uropods that stainedpositive for DiIC16 and HIV-1.

HIV-1 proteins were detected in isolated lipid raft fractions. Lipidrafts were purified by cell lysis and equilibrium centrifugation inorder to confirm the presence of HIV-1 proteins in these membranestructures. The fractions were assayed for the presence of viral andhost proteins by immunoblot analysis. The separation ofdetergent-resistant lipid rafts was confirmed by the abundance of Thy-1and CD59 in fractions 3 through 5, while CD45 was present only in thebottom fractions 9 and 10 (see Nguyen and Hildreth, supra, 2000; FIG.6). Immunoblot detection of membrane fractions revealed that the HIV MAprotein, p17, and gp41 were both present in the detergent-insolublelipid rafts of infected cells. The distribution between GEM domains andsoluble fractions was quantitated by MacBAS software version 2.5. Sincelysates were prepared from whole cells, the anti-MA MAb could bind notonly the membrane associated MA protein, but alsonon-membrane-associated forms of the group-specific antigen precursorprotein (Gag) and MA, thus accounting for the abundance of p17 detectedin the soluble fractions of the blot.

A possible mechanism for the targeting of the HIV Env protein, gp41, tolipid rafts involves palmitylation of two cysteines in its cytoplasmictail (see Yang et al., Proc. Natl. Acad. Sci., USA 92:9871-9875, 1995).Dual acylation of host proteins involving palmitate and myristate targetproteins to lipid rafts (Robbins et al., supra, 1995). As expected, asubstantial portion of the transmembrane subunit of Env, gp41, also waspresent in the GEM fractions. Theses results indicate that palmitylatedgp41 partitions specifically to lipid rafts. Since palmitylation is areversible post-translational modification, it is not likely that all ofthe gp41 present in the cell is palmitylated at any given time. Thiscould account for the large proportion present in soluble domains. Othertransmembrane proteins, such as MHC I and CD63, previously shown to beincorporated into HIV-1, were detected in lipid rafts as well, althoughthe majority of both molecules are in the solubilized fractions

Myristylated HIV-1 Gag partitioned to GEM domains. The myristylation ofGag is necessary for membrane association, proteolytic processing, andvirus budding. As such, it may be expected that, with a mixed populationof myristylated and non-myristylated Gag within a cell, only themyristylated forms will be responsible for membrane association andpotentially determining the site of virus budding. To ensure thatcellular Gag and not virus-associated Gag was being examined, a MAbspecific for p24 and p55 was used. To determine which areas of themembrane myristylated Gag would bind, cells were labeled with(³H)-myristic acid and isolated lipid rafts were examined. Lipid raftfractions and soluble fractions were pooled separately andimmunoprecipitated with an anti-Gag MAb. Blotting of the precipitatedproteins showed that myristylated Gag protein was present predominantlyin the lipid raft fractions (see Nguyen and Hildreth, supra, 2000; FIG.7). The blots were quantitated by densitometry, and the IgG backgroundswere subtracted from each to determine the distribution of the myristicacid label expressed as a percentage of the total. More than 90% of thecellular myristylated Gag was in lipid rafts. This result is consistentwith the observation that myristylation targets a number of hostproteins, such as Src, Lck, Lyn, and HCK protein tyrosine kinases, tolipid rafts as well.

In summary, these results demonstrate that HIV-1 budding occurs throughlipid rafts, thus accounting for the cholesterol-rich, sphingolipid-richvirus membrane, which bears GPI-linked proteins such as Thy-1 and CD59,and for the lack of CD45, which is not associated with lipid rafts incells.

EXAMPLE 2 Host Membrane Cholesterol is Required For HIV-1 Infection

This example demonstrates that intact lipid rafts and cholesterol arerequired for HIV-1 infection and syncytium formation.

Cells and Reagents

Jurkat, PM 1, and CEM×174 cell lines were obtained from the AmericanType Culture Collection (Rockville Md.) and maintained in cRPMI asdescribed in Example 1. Peripheral blood mononuclear cells were isolatedfrom leukopheresis buffy coats and stimulated with PHA as previouslydescribed (Gomez and Hildreth, supra, 1995). Control mouse myeloma IgG1and rabbit anti-mouse IgG (Fc specific) were purchased from JacksonImmunoresearch (West Grove Pa.). MAbs to MHC class I antigen (MHM.5),MHC class II antigen (MHM.33), CD4 (SIM.4), CXCR4 (FSN.NT.M3) and CD45(H5A5) were produced as described in Example 1.

2-OH-βCD Treatment and Virus Production

PM1 cells chronically infected with HIV-1_(RF) were washed and treatedwith 20 mM 2-OH-βCD in cRPMI or with cRPMI alone for 1 hr at 37° C. Thecells were then washed twice before resuspension at a density of5×10⁶/ml in cRPMI. The cells were incubated for 6 hrs at 37° C., 5% CO₂and pelleted by centrifugation. Supernatants, which contained the virus,were collected and purified by centrifugation through a 20% sucrosecushion (Liao et al., supra, 2000). The virus pellets were taken up incRPMI and titrated against LuSIV cells to determine infectivity. P24 wasmeasured in a standard p24 ELISA.

Cholesterol Measurement

Cellular cholesterol was measured with a sensitive cholesteroloxidase-based fluorimetric assay (Amplex Red Cholesterol Kit) fromMolecular Probes (Eugene Oreg.). Cholesterol content of cells wasnormalized to total cellular protein.

Syncytium Assays

Syncytium assays were carried out essentially as previously described(Hildreth and Orentas, supra, 1989). Briefly, cell lines or PHA blastswere treated with 20 mM 2-OH-βCD in RPMI 1640 or medium alone for 1 hrat 37° C. before washing twice with PBS. The treated cells were thenmixed with HIV-1 infected cells, each at density of 2×10⁶/ml, in cRPMIand incubated at 37° C. Syncytia were scored and photographed 3 to 6 hrafter mixing. For free-virus mediated syncytium assays (“fusion fromwithout”), HIV-1_(RF) from infected PM1 culture supernatants wereclarified by 0.45 Tm filtration (p24 concentration greater than 500ng/ml). 2-OH-βCD-treated and non-treated cell lines were added to thevirus preparations and incubated at 37° C. for 3 hr before countingsyncytia.

Flow Cytometry

Flow cytometry was performed as previously described (Orentas andHildreth, supra, 1993). Briefly, 2×10⁵ 2-OH-βCD treated or untreatedcells in 100 μl PBS containing 5% normal goat serum (NGS) were added to100 μl of MAb (1-5 μg) and incubated 30 min on ice. In comparativeexperiments we prefixed the cells with 2% paraformaldehyde in PBSimmediately after 2-OH-βCD treatment. Cells were washed with PBS,resuspended in 100 μl of PBS, 5% NGS containing 2 Tg of FITCconjugated-goat anti-mouse IgG (FITC-GAM) and incubated 1 h on ice.Cells were then washed with PBS and fixed with 2% paraformaldehydefollowed by analysis on an EPICS Profile II flow cytometer.

Confocal Microscopy

Cell-surface staining of 2-OH-βCD-treated and untreated cells wasperformed under saturating conditions. 2-OH-βCD-treated and untreatedPHA blasts (3×10⁵) were washed in cold PBS and pre-incubated on ice for15 min in 5% NGS/PBS. Cells were then incubated with MAb 1-5 μg in 5%NGS/PBS 45 min on ice, washed with PBS and then incubated with 2 μg ofFITC-GAM in 5% NGS/PBS. The cells were then fixed with 2%paraformaldehyde in PBS and spun onto poly-L-lysine coated slides usingcyto-funnels. The pellets were overlaid with 50 μl of 25% glycerol inPBS and a coverslip was positioned over the droplet. The edges of theslides were sealed with nail polish before storing them at 4° C. Thisstaining procedure was also performed with cells prefixed with 2%paraformaldehyde in PBS prior to MAb staining. Viewing of slides wasperformed with an Olympus IX50 confocal microscope under oil immersionat 100x magnification. Micrographs were acquired onto a Silicon GraphicsWorkstation with Intervision software. Final images were enhanced on theSilicon Graphics Workstation by two-dimensional deconvolution, andbrightness and contrast were adjusted for viewing.

Free Virus Binding Assay

Virus binding was measured through host cell antigen transfer asdescribed (Liao et al., supra, 2000). Briefly, Jurkat cells (1×10⁶) werewashed with serum free RPMI-1640 medium (iRPMI) before incubation in 20mM 2-OH-βCD in iRPMI or iRPMI alone for 1 hr at 37° C. Cells were thenwashed twice with iRPMI before adding 100 Tl of clarified HIV-1supernatant (>10 ng/ml of p24 from PM1 cells) for 1 hr on ice. Excessvirus was removed by washing twice with iRPMI. MAbs were then added at20 ug/ml in 5% NGS/PBS and allowed to incubate for 1 h on ice beforewashing with iRPMI. FITC-GAM (10 ug/ml) was then added for 45 min on icebefore washing with iRPMI. Cells were then fixed with 2%paraformaldehyde followed by analysis on an EPICS Profile II flowcytometer.

Primary Virus Infection Assay

Peripheral blood mononuclear cells (PBMCS) were isolated byFicoll-Hypaque centrifugation from buffy coats obtained from the JohnsHopkins Hemapheresis Center. Cells were stimulated for 3 days with 3Tg/ml PHA, washed with iRPMI, and treated with 10 mM 2-OH-βCD in cRPMIfor 1 hr. Cells were then washed twice with iRPMI and resuspended incRPMI (1×10⁶/ml) supplemented with 50 U/ml IL-2 and containing primaryHIV-1 strains at 20 ng/ml p24. Cells were incubated with virus for 24 hrat 37° C. before washing twice with iRPMI. Cells were then resuspendedin cRPMI supplemented with 50 U/ml IL-2 and cultured for 6 days at 37°C. Supernatants were collected and p24 was quantitated by standard p24ELISA.

Luciferase-based Infectivity Assay

The effect of 2-OH-βCD on infectivity of HIV was measured in aLuciferase-based single cycle infection assay as previously described(Liao et al., supra, 2000). LuSIV cells were treated with 20 mM 2-OH-βCDin iRPMI or iRPMI alone for 1 hr at 37° C. Cells were then washed withiRPMI before being resuspended in cRPMI at a density of 2×10⁶/ml. Cellsin 100 Tl were mixed with cRPMI or with dilutions of virus supernatant(62 to 500 pg/ml of p24) from 2-OH-βCD-treated or untreated PM1 cellsand allowed to incubate overnight (16 hr) at 37° C. The LuSIV cells werewashed with PBS and lysed with 100 Tl of Reporter Lysis Buffer(Promega). After centrifugation at 13,000×g for 30 seconds, 10, μl oflysate were added to 100 μl Luciferase Reagent (Promega) in an opaque 96well plate and luminescence was measured on a Packard Lumicountluminometer (Downers Grove, Ill.).

SDF-1α-Induced Cell Adhesion Assay

Cell adhesion assays were carried out essentially as previouslydescribed (Liao et al., supra, 2000). The wells of 96 well plates werecoated with recombinant ICAM-Ig and blocked as described. Jurkat cellswere labeled with horseradish peroxidase (HRP) and treated with either20 mM 2-OH-βCD or medium alone as described above. The cells were thenwashed and resuspended in cRPMI at a density of 2×10⁶/ml. One hundred Tlof cells were added to the wells along with cRPMI alone or mediumcontaining 10 ng/ml of SDF-1α. The wells were incubated at 37° C. forvarious times before washing to remove unbound cells. Bound cells werelysed and HRP measured as described previously. A standard curve wasgenerated by from known numbers of labeled cells lysed and quantitatedby measuring HRP.

Results

2-OH-βCD treatment blocked syncytium formation of primary cells and celllines. The role of lipid rafts in the HIV-1 fusion process was examinedby treating CD4+HIV-susceptible target cells with 2-OH-βCD to depletemembrane cholesterol and disperse lipid rafts. Treatment of cells with10 to 20 mM 2-OH-βCD for 1 hr at 37° C., followed by washing to removefree 2-OH-βCD, depleted greater than 70% of total cellular cholesterolwithout any loss in cell viability as measured by Trypan Blue exclusion.Furthermore, treated cells continued to grow normally after 2-OH-βCDtreatment when placed back into culture in cholesterol-containingmedium. The non-toxicity of βCD treatment was further demonstrated byfinding 2-OH-βCD treated Jurkat cells still showed Ca²⁺ flux responsesto anti-CD3 MAb.

CD4+SupTl T cells formed numerous large syncytia within 3 hr after theaddition of HIV-1_(MN)-infected H9 cells. 2-OH-βCD treatment of SupTlcells completely inhibited syncytium formation with HIV-1_(MN)-infectedH9 cells. No syncytia were apparent in this culture for more than 15 hr,which can reflect the recovery time for cholesterol in the βCD treated Tcells. 2-OH-βCD was washed out after the 1 hr treatment and was notpresent during the co-cultivation step. As such, the effects are not aresult of a steric blockade by 2-OH-βCD, which can bind to cells.

To confirm that the effects of the βCD on syncytium formation were dueto cholesterol depletion, cells were treated with 2-OH-βCD that had beenpre-loaded (saturated) with cholesterol (CH-βCD) and, therefore, wasunable to deplete cellular cholesterol. Cells treated with the CH-βCDfused to HIV-1-infected cells as efficiently as control untreated cells,thus confirming that the βCD blocked HIV-induced fusion by depletingcholesterol. Similar results were obtained when primary cells (PHAstimulated T cells) that had been treated with 2-OH-βCD or CH-βCD wereused as fusion partners with HIV-infected cells.

The effect of βCD treatment on HIV-induced fusion of several other celllines also was examined. Four CD4+, CXCR4+cell lines, SupT1, H9, PM1,and MT2, were treated with 2-OH-βCD and tested for syncytium formationwith HIV-1-infected cells. In each case, syncytium formation wascompletely blocked by depleting cellular cholesterol (see Table 1).These results demonstrate that cholesterol and intact lipid rafts arerequired for HIV-induced syncytium formation.

TABLE 1 2-OH-βCD Effects on CD4 and CXCR4 Surface Expression andSyncytium Formation of Cell Lines with HIV-infected H9 Cells SyncytiaCD4 CXCR4 (/HPF) (MCF) (MCF) Cell Line Control βCD Control βCD ControlβCD MT2  55 ± 10 0 65.3 62.2 30.1 16.7 PM1 71 ± 5 0 78.6 85.9 16.2 3.1H9 52 ± 3 0 21.2 20.9 29.9 6.7 SupT1 63 ± 8 0 140.1 188.3 44.9 15.0“MCF” indicates mean channel fluorescence. “βCD” indicates treatmentwith 20 mM 2-OH-βCD in medium.

The effect of cholesterol depletion on virus-cell fusion (“fusion fromwithout”) was also determined. CD4+/CXCR4+cell lines (MT2, SupT1, PM1)incubated with free HIV-1_(RF) at concentrations greater than 500 ng/mlof p24 for 3 hr at 37° C. showed extensive syncytium formation (>30syncytia per HPF), whereas cells treated with 2-OH-βCD showed nosyncytium formation when exposed to virus under the same conditions.Although low levels of fusion that do not proceed to gross syncytiacannot be detected, these results indicate that cholesterol depletionblocked HIV-induced cell-cell fusion from without, which first requiresextensive virus-cell fusion to put HIV envelope proteins into cellmembranes. These results indicate that cholesterol depletion preventsfusion of HIV particles to cells.

Cholesterol depletion also promoted CXCR4 down-modulation by MAb-inducedinternalization. A possible explanation for the inhibition of syncytiumformation by cholesterol depletion as described above is that CD4, CRs,or both are lost from the cell surface, for example, by extrusion fromthe membrane in vesicles after loss of cholesterol, or they could beinternalized. To explore these possibilities, βCD treated cells wereexamined for expression of HIV receptors by flow cytometry. CD4expression did not change after treatment with 2-OH-βCD in PHA blasts orany of the cell lines tested (Table 1). In contrast, cell surfaceexpression of CXCR4 was reduced by 50% or more in all of the cells after2-OH-βCD treatment (Table 1). PM1 cells showed the most significant lossof CXCR4 expression, with a drop in total mean channel fluorescence(MCF) from 16.2 to 3.1. Primary T cells showed a similar reduction inCCR5 expression, from 19% to 8% of cells staining positive.

In order to determine whether the loss of CXCR4 expression was due toMAb-induced internalization, cells were fixed with 2% paraformaldehydein PBS immediately after 2-OH-βCD treatment and before staining withMAbs for flow cytometry. Under these conditions both CD4 and CXCR4expression remained unchanged on the cell surface (Table 2).2-OH-βCD-treated cells fixed and permeabilized after the MAb bindingstep showed no significant reduction in anti-CXCR4 MAb staining comparedto control cells. These results demonstrate that CXCR4 remains on thesurface after βCD treatment, but is rapidly internalized following MAbbinding.

TABLE 2 2-OH-βCD Treatment Does Not Down modulate CXCR4 CD4 (MCF) CXCR4(MCF) Cell line Control βCD Control βCD PM1 146 129 63 64 H9 46 50 45 55SupT1 216 213 94 91 Cells were fixed with 2% paraformaldehyde in PBSimmediately after βCD treatment before performing flow cytometry. “MCF”indicates channel fluorescence; 2-OH-βCD treatment (20 mM).

Immunostaining and confocal microscopy of βCD-treated PHA blasts andcontrol cells showed that CXCR4 was not significantly redistributed onthe cell surface after cholesterol depletion. Patchy staining of CXCR4persisted after 2-OH-βCD treatment whether the cells were fixed beforeor after mAb staining. Consistent with flow cytometry data, overallstaining was reduced in the βCD-treated cells that were not fixed beforethe MAb staining procedure. The distribution of CD4 and CD45 wasunchanged on the cell surface after 2-OH-βCD treatment. These resultsdemonstrate that the overall membrane expression and distribution ofcritical HIV receptors were essentially unchanged after βCD treatmentand suggest that the cholesterol content of the cell membrane is acritical factor in HIV-induced membrane fusion.

βCD treatment reduced HIV-1 binding. The possibility that βCD treatmentaffected virus binding or interactions between gp120 and CD4 or CRs wasexamined. In order to measure virus binding to cells, a flow cytometryassay was used that measures the transfer of host cell class II MHCproteins to class II MHC-negative cells by HIV virions, whichincorporate large numbers of these proteins into their lipid envelopes.This approach previously was used to demonstrate adhesionmolecule-mediated binding of HIV to cells (Liao et al., supra, 2000).Class II MHC-negative Jurkat cells were used as target cells in the HIVbinding assay. As a positive control for flow cytometry analysis, classI MHC molecules were probed on the Jurkat cells, which were stainedequally well with MAb against this protein before and after 2-OH-βCDtreatment (Table 3). When HIV-1_(RF) from class II MHC-positive PM1cells was added to untreated Jurkat cells, class II MHC MAb mean channelfluorescence increased from 1.0 to 7.8 relative fluorescence units,while the percentage of positive cells increased from 3.0% to 55.4%(Table 3). 2-OH-βCD treatment of the cells reduced virus binding by 70%,as determined by mean channel fluorescence of the anti-class II MHC mAb.These results demonstrate that HIV-1 remains capable of measurableattachment after cholesterol depletion of target cells, but at muchlower levels.

TABLE 3 2-OH-βCD Reduces Binding of HIV-1 to Target Cells IgG1 MHM5MHM33 MCF (% Pos) MCF (% Pos) MCF (% Pos) Control βCD Control βCDControl βCD No virus 0.9 (2.4) 0.7 (3.4) 158.6 (100)   165 (100) 1.0(3.0)  0.7 (3.3)  HIV_(RF) ND ND    164 (99.9) 167.8 (100) 7.8 (55.4)2.4 (26.0)HIV binding to Jurkat cells (control and 2-OH-βCD-treated) was measuredby transfer of class II MHC molecules. MAbs used were MHM.5 (anti-classI MHC), positive control; MHM.33 (anti-class II MHC). “MCF” indicatesmean channel fluorescence. “% Pos” indicates percent positive cells.“ND” indicates not determined.

2-OH-βCD treatment blocked CR-induced LFA-1 function. The resultsdescribed above suggested that intact lipid rafts were required forstable membrane expression of CXCR4. Loss of CXCR function in regulatingLFA-1 could also explain the lower binding of HIV-1 to βCD-treated cellsobserved previously (Liao et al., supra, 2000; Orentas and Hildreth,supra, 1993). As before, the treatment with βCD had no effect on cellviability. The ability of βCD treatment of cells to affect control ofLFA-1 function by CXCR4 also was examined. Jurkat cells were treatedwith 2-OH-βCD or medium alone, then were added to the wells of cultureplates coated with soluble recombinant ICAM-Ig. SDF-1, a CXCR4-specificchemokine that triggers LFA-1 function, was added to trigger binding ofLFA-1 to ICAM-1. Control cells responded to SDF-1 and, as expected,bound very well to ICAM-Ig. In contrast, the βCD-treated Jurkat cellsshowed no binding to ICAM-Ig after exposure to SDF-1. These results areconsistent with previous reports showing disruption of integrin functionby cholesterol depletion. CXCR4-specific gp120 triggers the sameresponses through CXCR4 as SDF-1 (Iyengar et al., J. Immunol.162:6263-6267, 1999). These results indicate that HIV-1 particles cannottrigger LFA-1 function on βCD-treated cells, which may explain lowervirus binding to such cells.

βCD treatment blocks HIV-1 virus infection. HIV-1 can spread in cellcultures without necessarily exerting cytopathic effects. Thus,inhibition of syncytium formation by cholesterol depletion and lipidraft dispersion does not necessarily mean that HIV infection by freevirus also is blocked. To test the effects of cholesterol depletion onHIV-1 infection of primary T cells by free virus, PHA blasts weretreated with 10 mM 2-OH-βCD or medium alone, then were exposed to HIV-1for 2 hr before washing to remove input virus. Viability and growth ofthe PHA blasts was not affected by treatment with the βCD under theconditions used. P24 release was measured after an addition 6 days inculture. Two primary strains of HIV-1, 97.099 and 97.534, M-tropic (R5)and dual-tropic (X4R5), respectively, were tested. The results wereidentical to those obtained in syncytium formation assays; 2-OH-βCDtreatment of PHA blasts completely inhibited infection by HIV isolate97.099, while infection by isolate 97.534 was inhibited by more than70%.

The effects of βCD treatment on HIV infectivity were measured in asensitive single-cycle infection assay based on a cell line transfectedwith an LTR-luciferase cassette. The CD4+CEM×174 (LuSIV) cells possess amodified SIV LTR viral promoter linked to the luciferase gene.Quantitative measurements of single round infection are obtained withthis assay system (Roos et al., Virology 273:307-315, 2000, which isincorporated herein by reference). Viability of LuSIV cells asdetermined by trypan blue exclusion and proliferation was not affectedby 2-OH-βCD treatment. βCD treatment of LuSIV cells reduced HIVinfection by almost 100%, and the effects were readily seen at all viralinput levels. The effect of the βCD treatment of LuSIV cells on HIVinfection was completely reversed by exposing the 2-OH-βCD-treated cellsto CH—BCD (48 Tg/ml cholesterol) for 1 hr to restore membranecholesterol before exposing the cells to HIV. These results demonstratethat cholesterol in the membrane of HIV susceptible cells is requiredfor infection by free virus.

EXAMPLE 3 β-Cyclodextrin Blocks Vaginal Transmission of Cell-AssociatedHIV-1

These results demonstrate that topical administration of a βCD reducestransmission of cell-associated HIV-1 through vaginal epithelium.

Cell Culture

Blood was obtained from HIV-negative volunteers by the Johns HopkinsUniversity Hemapheresis Laboratory. HuPBMC were isolated usingFicoll-Hypaque (Pharmacia; Uppsala, Sweden) and were washed andsuspended at 5×10⁷/ml in PBS prior to intraperitoneal administration toSCID mice. HuPBMC that were used as inocula were maintained in cRPMI(RPMI-1640 supplemented with 10% FCS, penicillin, streptomycin andgentamycin. PBMC were stimulated with PHA (Sigma) for 2 days; cells wereexposed to 300 TCID₅₀ (50% tissue culture infective dose) ofHIV-1_(Ba-L) in cRPMI with IL-2 (10 U/ml, Boehringer Mannheim).Infected-cell cultures were maintained in cRPMI with IL-2 for 10 daysprior to inoculation into the mice.

Limiting dilution-PCR was performed using HIV-1 gag-specific primers asdescribed previously (Markham et al., Proc. Natl. Acad. Sci., USA95:12568-12573, 1998, which is incorporated herein by reference) todetermine the extent to which HuPBMC were infected with HIV-1. To assesvirus recovery from cells harvested from the peritoneal cavities ofchallenged mice, uninfected HuPBMC were PHA-stimulated and maintained inIL-2-supplemented media (1×10⁶/mouse) in preparation for co-culture withperitoneal cells recovered from the HuPBL-SCID mice.

HIV-1 Virus Preparation

A single lot of the inoculum virus, HIV-1_(Ba-L), was purchased (ABI,Inc.; Columbia Md.), aliquoted and stored in liquid N₂ until used toinfect HuPBMC. The TCID₅₀ inoculated was confirmed using MAGI cells(CD4, CCR5, and HIV-LTR-βgal-transfected HeLa), and by titration onperipheral blood-derived monocytes.

Vaginal Infection of HuPBL-SCID Mice with HIV-1

Female mice with severe combined immunodeficiency (C.B-17scid; Bosma etal., Nature 301:527-530, 1983; Bosma and Carroll, Ann. Rev. Immunol.9:323-350, 1991, each of which is incorporated herein by reference),were obtained from Charles River Laboratories (Wilmington Mass.) or froma SCID mouse colony established using C.B-17 mice from JacksonLaboratories (Bar Harbor Me.).

The mice were treated subcutaneously with 2.5 mg progestin(Depo-Provera® progestin; Upjohn Pharmaceuticals; Kalamazoo Mich.), onthe same day as administration of 5×10⁷ unstimulated HuPBMCintraperitoneally in 1 ml PBS. Seven days following progestin treatmentand reconstitution of the SCID mice with HuPBMC, the mice wereanesthetized and administered pelleted, cell-free HIV-1_(Ba-L) (up to106 TCID₅₀), supernatant fluids from HIV-1_(Ba-L)-infected HuPBL,HIV-1_(Ba-L)-infected HuPBL, or HIV-1_(MN)-infected HuPBL (1×10⁶/mouse).In the βCD experiments, the mice received 2-OH-βCD (3% w/v in PBS) 5 minprior to receiving 1×10⁶ HIV-1_(Ba-L)-infected HuPBL, 1×10⁶HIV-1_(Ba-L)-infected HuPBL pre-incubated with 3% 2-OH-βCD, or 1×10⁶HIV-1_(Ba-L)-infected HuPBL suspended in PBS.

Mice remained anesthetized for 5 minutes following intravaginalinoculation by pipette. Extreme care was taken to avoid causing traumato vaginal tissues. Two weeks later the mice were euthanized andperitoneal cells were recovered by lavage with cold PBS. The cellsrecovered by lavage (of both murine and human origin) were assayed byDNA-PCR for human β-globin to determine the presence of human cells fromthe peritoneum and for HIV-1 infection by co-culture with PHA-stimulatedHuPBMC.

Vaginal Epithelial Morphology

Four BALB/c and four HuPBL-SCID mice were sham-treated or treated with2.5 mg Depo-Provera progestin one week prior to the experiment. Micewere euthanized by cervical dislocation and reproductive tissues werecollected and dissected. Excised vaginal tissue was fixed (Omnifix;Zymed Laboratories; San Francisco Calif.) overnight and embedded inparaffin, sectioned and stained with hematoxylin and eosin.

Fluorescent In Situ Hybridization (FISH)

Six SCID mice were treated with 2.5 mg progestin, with or without HuPBLreconstitution (i.e., peritoneal transplant of human cells). Spleen withperitoneal mesentery, and vaginal tissues were fixed inparaformaldehyde, and embedded in paraffin. Sections were mounted onslides, de-paraffinized, and made permeable by immersion in 50% glycerolin 0.1X SSC at 90° C., followed by incubation in protease solution(Hyytinen et al., Cytometry 16:93-99, 1994, which is incorporated hereinby reference). Sections were then co-denatured with a biotin-labeled,human pan-centromere probe (Cytocell; Oxfordshire, UK) at 75° C., andhybridized overnight. Slides were washed, and bound probe was detectedwith CY3-conjugated streptavidin (Cytocell). Tissue sections were DAPIcounterstained, and examined with epifluorescence microscopy.

Migration of Vaginally-Inoculated Human PBMC

HuPBMC were labeled with bisbenzamide (3 Tg/ml, Sigma); the fluorescenthuman cells (1×10⁷) were added vaginally to Depo-Provera®progestin-treated mice (7 SCID mice and 6 BALB/c), and 4 hr later theiliac lymph nodes of each mouse were removed and homogenized on a cellstrainer. Fluorescent (and non-fluorescent) cells were counted byfluorescence and phase-contrast microscopy.

Vaginal Epithelial Toxicity

CF-1 mice were pretreated with 2.5 mg progestin; one week later, threegroups of three mice each were inoculated with 50 Tl of either PBS, 1%(w/v) nonoxynol-9, or 3% (w/v) 2-OH-βCD (20 mM). Each test solution alsocontained the membrane-impermeant DNA binding fluorescent dye, ethidiumbromide homodimer-1 (20 TM, Molecular Probes; Eugene Oreg.; Hyytinen etal., supra, 1994). Fifteen min following exposure to the test agents themice were euthanized, the vaginas were dissected and openedlongitudinally, and viewed using a fluorescent microscope and TRITCfilter set.

Effect of Progesterone Treatment on Murine Vaginal Mucosa

Studies in non-human primate models of simian immunodeficiency virus(SIV) or chimeric simian-human immunodeficiency virus (SHIV)transmission have frequently pre-treated the challenged animals withprogesterone, with the stated purpose of synchronizing the estrous cycleamong experimental animals. However, this treatment also thins thevaginal epithelium, facilitating viral transmission by this route (see,for example, Sodora et al., AIDS Res. Hum. Retrovir.14(Suppl.1):S119-123, 1998). Progestin treatment of HuPBL-SCID micesimilarly caused the multi-layer, stratified, squamous epithelium of theuntreated mouse vagina to assume a cervix-like, single layer, columnarmorphology. Thus progestin treatment had the effect of rendering themouse vagina morphologically unlike the human vagina, and more similarto the human, columnar cervical epithelium, which in organ culture ismore readily infected with HIV-1 than is vaginal tissue (Howell et al.,J. Virol. 71:3498-3506, 1997). In a series of pilot studies, neithercell-free nor cell-associated HIV-1 could be transmitted in HuPBL-SCIDmice by the vaginal route without prior administration of progestin(medroxyprogesterone acetate, Depo-Provera® progestin).

HuPBL-SCID Mice Susceptible to Vaginal Transmission of Cell-AssociatedHIV-1 but not to Cell-Free HIV-1

To determine whether HuPBL-SCID mice were susceptible to cell-associatedor cell-free virus, mice were exposed vaginally to cell-freeHIV-1_(Ba-L) (CCR-5-utilizing strain) and HIV-1_(Ba-L) infected humanperipheral blood mononuclear cells (HuPBMC; see Table 4.

TABLE 4 Vaginal Transmission of Cell-Associated HIV-1 in HuPBL-SCIDmice^(a) Number of mice from which HIV-1 was cultured/ HIV inoculumNumber of mice exposed to HIV-1 HIV-1_(Ba-L)-infected HuPBMC 1.00 × 10⁶cells  5/5* 0.25 × 10⁶ cells  4/5* 0.05 × 10⁶ cells 1/5 HIV-1_(Ba-L)cell-free virus 1.0 × 10⁶ TCID₅₀ 0/5 1.0 × 10⁵ TCID₅₀ 0/5 P = 0.048compared with cell-free virus. ^(a)Representative of at least 3 separateexperiments of ≧5 mice per group.

High-titer, cell-free HIV-1 and virus obtained from supernatant fluidfrom the HIV-1 infected HuPBMC used in the same experiment (i.e.,recently-budded virus) also were tested. HIV-1 infected HuPBMC wereprepared for inoculation into the mice 10 days after in vitro exposureof PHA- and IL-2-stimulated cells to HIV-1. Despite intravaginalinoculation of up to 1×10⁶ TCID₅₀ of cell-free HIV-1_(Ba-L), virus wasnot transmitted to the HuPBMC that were transplanted intraperitoneallyin the mice. However, cell-associated HIV-1_(Ba-L)was efficientlytransmitted vaginally in the HuPBL-SCID mice with as few as 250,000HuPBMC, between 1% and 5% of which were infected with HIV-1 (i.e. as fewas 10^(3.5) HIV-1-infected cells). HuPBMC infected to similar levelswith HIV_(MN), a CXCR4-using variant, and inoculated intravaginally,transmitted infection much less efficiently (Table 5).

TABLE 5 Vaginal Transmission of Cell-Associated R5-utilizing HIV-1^(a)Number of mice from which HIV-1 was cultured/ HIV inoculum Number ofmice exposed to HIV-1 HIV-1_(Ba-L)(R5)-infected HuPBMC 1.00 × 10⁶ cells 5/5* 0.25 × 10⁶ cells  4/5* 0.05 × 10⁶ cells 1/5HIV-1_(MN)(X4)-infected HuPBMC 1.00 × 10⁶ cells 1/5 0.25 × 10⁶ cells 0/50.05 × 10⁶ cells 0/5 *P = 0.048 compared to an equal number ofHIV-1_(MN)-infected HuPBL. ^(a)Representative of at least 2separateexperiments with ≧5 mice per group.Human PBMC Transplanted into the Peritoneal Cavity of HuPBL-SCID Mice DoNot Populate the Reproductive Tract

To determine if human cells placed into the peritoneal cavity couldmigrate to sites in the vaginal mucosa and/or submucosa, HuPBMC weretransplanted into the peritoneal cavities of progesterone-treated,female HuPBL-SCID mice. Seven days later the mice were euthanized andtissue sections of the vagina, spleen and peritoneal mesentery werehybridized with a human pan-centromere probe to detect human cells.Whereas abundant human cells were found in the peritoneal mesentery, andoccasionally in the spleen of all the HuPBL-SCID mice, no human cellswere detected in the vaginal tissues. Thus, there do not appear to beany locally accessible target cells in the vagina, which free virus caninfect following intravaginal inoculation, in these mice.

Vaginally Introduced Human PBMC Migrate to Regional Lymph Nodes ofHuPBL-SCID Mice

To define the basis for transmission of cell-associated virus, infectedand uninfected HuPBMC were examined for the ability to migrate from thevagina to the site of transplanted human cells in the peritoneal cavity.HuPBMC were labeled with bisbenzamide (3 Tg/ml), then the fluorescenthuman cells (1×10⁷) were added vaginally to Depo-Provera®progestin-treated mice. Four hr later, the iliac lymph nodes of eachmouse were removed and homogenized on a cell strainer. Fluorescent cellswere detected in the lymph nodes (mean cell number 204, ranging from0-395, up to 5% of the cells recovered from the lymph nodes of SCIDmice). The human cells migrated to the iliac lymph nodes of bothprogestin-treated BALB/c and progestin-treated HuPBL-SCID mice.

2-OH-βCD Prevents Cell-Associated HIV-1 Transmission and is Non-Toxic tothe Vaginal Epithelium

The ability of a βCD to inhibit vaginal transmission of cell-associatedHIV-1 was examined. Reconstituted HuPBL-SCID mice were challengedintravaginally with 1×10⁶ PBMC infected with HIV_(Ba-L) after receivingprogesterone subcutaneously and HuPBMC intraperitoneally. For two of theexperimental groups the HIV-1 infected PBMC were pre-incubated in either20 Tl PBS or 2-OH-βCD (3% w/v) before the mixture was inoculatedintravaginally. A third group of mice received 20 Tl 2-OH-βCD (3% w/v)intravaginally, followed 5 min later by the infected PBMC in PBS (10Tl). βCD significantly inhibited cell-associated HIV-1 transmission bythis route, even when administered prior to exposure to HIV-1 infectedPBMC (Table 6).

TABLE 6 2-OH-βCD Inhibits Vaginal Transmission of Cell-Associated HIV-1Number of mice from which HIV-1 was cultured/ Treatment of HIV-infectedcells Number of mice exposed to HIV-1 PBS premixed with HuPBMC 12/17 2-OH-βCD premixed with 2/16* HuPBMC 2-OH-βCD administered 1/11*intravaginally prior to infected HuPBMC challenge *P < 0.01 compared toPBS premixed with HuPBMC.

To examine the effect of a βCD on the vaginal epithelium, CF-1 mice werepretreated with progesterone and one week later were inoculated with 50Tl of either phosphate buffered saline (PBS), 1% (w/v) nonoxynol-9, or3% (w/v) 2-OH-βCD (20 mM) containing the membrane impermeant DNA bindingfluorescent dye, ethidium bromide homodimer-1. The vaginas of the micewere viewed by fluorescent microscopy. 1% nonoxynol-9 causedconsiderable epithelial damage. In contrast, the 2-OH-βCD-treated micehad only minimal membrane damage to the vaginal epithelial cells andappeared more similar to the vaginal epithelium of the PBS treatedcontrol mice.

These results demonstrate that a βCD can inhibit vaginal transmission ofcell-associated HIV-1. In addition, the results demonstrate that theHuPBL-SCID mice are useful for examining the effectiveness of potentialagents that can reduce the sexual transmission of sexually transmitteddiseases.

EXAMPLE 4 β-Cyclodextrin Blocks Vaginal Transmission of HSV-2

This example demonstrates that a βCD can be used reduce the risk oftransmission of HSV-2 into vaginal epithelial cells.

The ability of 2-OH-βCD to reduce the infectiousness of HIV-1 (Examples2 and 3) led to an investigation as to the general applicability of βCDsto prevent the transmission of other sexually transmitted envelopedviruses. A well characterized HSV mouse vaginal challenge model was usedto examine the effect of 2-OH-βCD on HSV-2 infection (Sherwood et al.,supra, 1996). Adult female CD-1 mice (Charles River BreedingLaboratories; Wilmington Mass.) were injected subcutaneously withDepo-Provera® progestin (2 mg) to increase their sensitivity, then oneweek later the mice were inoculated intravaginally with 1×10⁴ TCID₅₀ ofcell-free HSV-2 strain G (10 ID₅₀) using a WIRETROL capillarymicropipette (Drummond Scientific; Broomall Pa.). Infection wasconfirmed by lavage culture on newborn foreskin fibroblast cells(Biowhitaker). Prior to inoculation, HSV-2 was preincubated either with3% 2-OH-βCD or with buffer.

Nine of nine mice inoculated with the HSV-2 that had been preincubatedwith buffer (positive control) became infected. In comparison, only 5 of10 mice inoculated with the βCD treated HSV-2 became infected (p=0.022).These results demonstrate that βCD can reduce the infectiousness ofHSV-2, and further demonstrate the general effectiveness of βCDs forreducing the risk of transmission of sexually transmitted pathogens,including sexually transmitted enveloped viruses.

EXAMPLE 5 Effectiveness of β-Cyclodetrin Against HSV-2

This example provides a method for determining the effectiveness ofintravaginal administration of βCD to reduce the risk of transmission ofHSV-2.

Adult female mice are treated with Depo-Provera® progestin as describedin Example 4. One week later, the mice are treated with 10 Tl of BartelsTissue Culture Refeeding Medium (Bartels; Issaquah Wash.) containing1×10⁴ TCID₅₀ of cell-free HSV-2 (see Example 4). In various groups ofmice, the HSV-2 is treated with a βCD prior to inoculation, or the βCDis administered intravaginally either in a buffer solution or in aBufferGel gel composition prior to inoculation with buffer treatedHSV-2. Appropriate controls include BufferGel gel, alone, or a differentgel compound.

Animals are observed daily for signs of infection, including perivaginalerythema, vesicles, and hair loss. Asymptomatic infection is detected byvaginal lavage and culture: 20 Tl of Bartels Tissue Culture RefeedingMedium is pipetted in and out of the vagina 10 times, diluted to 0.1 ml,and placed on target human newborn foreskin diploid fibroblast cells(Biowhitaker). Cytopathic effect (CPE) is scored 48 hr later, and thosewith lavage cultures displaying CPE are considered infected.

EXAMPLE 6 Effectiveness of β-Cyclodextrin Against Chlamydia

This example provides a method for determining the effectiveness ofintravaginal administration of βCD to reduce the risk of transmission ofa bacterial sexually transmitted pathogen, Chlamydia trachomatis.

A C. trachomatis mouse vaginal transmission model is used. Briefly, 6 to8 week old specific pathogen-free outbred CF-1 female mice arepretreated subcutaneously with DepoProvera® as described in Example 4.One week later, the mice are inoculated with C. trachomatis serovar D(“Ct-D”; ATCC Accession No. VR-885), which are propagated in McCoy cells(ATCC). On the day of inoculation, 50 Tl βCD (0, 1, 3 or 10% in saline)is delivered intravaginally using a 50 Tl WIRETROL pipette, and stirredto mimic the stirring effect of human coitus. Thirty min after deliveryof the βCD, 1×10⁵ inclusion forming units (ifu) of Ct-D (10 ID₅₀)suspended in 10 T1 of sucrose-phosphate transport medium is deposited inthe vagina using a 10 Tl WIRETROL pipette.

Transmission is detected by determining the presence of Ct-D in thelower genital tract by culture. Vaginal swabs (Type 1 DACROSWAB swabs;Spectrum Laboratories; Dallas Tex.) are taken from each mouse on days 4and 8 post-inoculation. Swabs are placed in transport medium and frozenin test tubes at ⁻80° C. Specimens are plated in duplicate onsemi-confluent McCoy cells, and the duplicate plate frozen following 72hr of incubation. After iodine staining and evaluation of the primaryplate, the duplicate plate is thawed and those specimens that arenegative on the primary culture are transferred onto fresh McCoy cellmonolayers for secondary (amplified) culture. Any positive cultureresult, either primary or secondary cultures on one or both days, isconsidered a productive infection.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for treating a subject infected with a pathogen comprising,administering a therapeutically effective amount of a compositioncomprising an active agent to the subject, wherein the active agent is aβ-cyclodextrin, thereby treating the subject.
 2. The method of claim 1,wherein the pathogen is a virus.
 3. The method of claim 2, wherein thevirus is an enveloped virus.
 4. The method of claim 3, wherein theenveloped virus is selected from the group consisting of a Tlymphotrophic virus, human immunodeficiency virus (HIV), herpes simplexvirus (HSV), and influenza virus.
 5. The method of claim 2, wherein thevirus is a human immunodeficiency virus.
 6. The method of claim 2,wherein the virus is a herpes simplex virus.
 7. The method of claim 2,wherein the virus is influenza virus.
 8. The method of claim 1, whereinthe pathogen is a microbial pathogen.
 9. The method of claim 8, whereinthe microbial pathogen is a bacterium, a yeast, or a protozoan.
 10. Themethod of claim 9, wherein the microbial pathogen is a Chlamydia spp., aTrichomona spp., or a Candida spp.
 11. The method of claim 1, whereinthe β-cyclodextrin is formulated in a solution, a gel, a foam, anointment, a cream, a paste, or a spray.
 12. The method of claim 1,wherein the β-cyclodextrin is formulated in a suppository, a film, acontraceptive, an agent for treating a sexually transmitted disease, alubricant, or a combination thereof.
 13. The method of claim 1, whereinthe composition further comprises an agent selected from the groupconsisting of a contraceptive, an agent for treating a sexuallytransmitted disease, a lubricant, and a combination thereof.
 14. Themethod of claim 12, wherein the contraceptive is selected from the groupconsisting of a diaphragm, a vaginal disk, a vaginal film, a sponge, aspermicide, and a condom.
 15. The method of claim 13, wherein thecontraceptive is selected from the group consisting of a diaphragm, avaginal disk, a vaginal film, a sponge, a spermicide, and a condom. 16.The method of claim 1, wherein the subject is a vertebrate.
 17. Themethod of claim 1, wherein the subject is a human.
 18. A method fortreating a subject infected with a pathogen comprising, administering atherapeutically effective amount of a composition consisting of aβ-cyclodextrin to the subject, thereby treating the subject.
 19. Amethod for treating a subject infected with a pathogen comprising,administering a therapeutically effective amount of a compositionconsisting of a β-cyclodextrin and at least one additional active agentother than β-cyclodextrin to the subject, thereby treating the subject.